I'm using .NET 4 and the new RegistryKey.FromHandle call so I can take the hKey I get from opening a registry file with RegLoadAppKey and operate on it with the existing managed API.

I thought at first it was just a matter of a busted DllImport and my call had an invalid type in the params or a missing MarshalAs or whatever, but looking at other registry functions and their DllImport declarations (for instance, on pinvoke.net), I don't see what else to try (I've had hKey returned as both int and IntPtr, both worked on 32-bit OS and fail on 64-bit OS)

I've got it down to as simple a repro case as I can - it just tries to create a 'random' subkey then write a value to it. It works fine on my Win7 x86 box and fails on Win7 x64 and 2008 R2 x64, even when it's still a 32-bit process, even run from a 32-bit cmd prompt. EDIT: It also fails in the same way if it's a 64-bit process.

on Win7 x86:

INFO: Running as Admin in 32-bit process on 32-bit OS
Was able to create Microsoft\Windows\CurrentVersion\RunOnceEx\a95b1bbf-7a04-4707-bcca-6aee6afbfab7 and write a value under it

on Win7 x64, as 32-bit:

INFO: Running as Admin in 32-bit process on 64-bit OS

Unhandled Exception: System.UnauthorizedAccessException: Access to the registry key '\Microsoft\Windows\CurrentVersion\RunOnceEx\ce6d5ff6-c3af-47f7-b3dc-c5a1b9a3cd22' is denied.
   at Microsoft.Win32.RegistryKey.Win32Error(Int32 errorCode, String str)
   at Microsoft.Win32.RegistryKey.CreateSubKeyInternal(String subkey, RegistryKeyPermissionCheck permissionCheck, Object registrySecurityObj, RegistryOptions registryOptions)
   at Microsoft.Win32.RegistryKey.CreateSubKey(String subkey)
   at LoadAppKeyAndModify.Program.Main(String[] args)

on Win7 x64, as 64-bit:

INFO: Running as Admin in 64-bit process on 64-bit OS

Unhandled Exception: System.UnauthorizedAccessException: Access to the registry key '\Microsoft\Windows\CurrentVersion\RunOnceEx\43bc857d-7d07-499c-8070-574d6732c130' is denied.
   at Microsoft.Win32.RegistryKey.Win32Error(Int32 errorCode, String str)
   at Microsoft.Win32.RegistryKey.CreateSubKeyInternal(String subkey, RegistryKeyPermissionCheck permissionCheck, Object registrySecurityObj, RegistryOptions registryOptions)
   at Microsoft.Win32.RegistryKey.CreateSubKey(String subkey, RegistryKeyPermissionCheck permissionCheck)
   at LoadAppKeyAndModify.Program.Main(String[] args)

source:

class Program
{
    static void Main(string[] args)
    {
        Console.WriteLine("INFO: Running as {0} in {1}-bit process on {2}-bit OS",
            new WindowsPrincipal(WindowsIdentity.GetCurrent()).IsInRole(WindowsBuiltInRole.Administrator) ? "Admin" : "Normal User",
            Environment.Is64BitProcess ? 64 : 32,
            Environment.Is64BitOperatingSystem ? 64 : 32);

        if (args.Length != 1)
        {
            throw new ApplicationException("Need 1 argument - path to the software hive file on disk");
        }
        string softwareHiveFile = Path.GetFullPath(args[0]);
        if (File.Exists(softwareHiveFile) == false)
        {
            throw new ApplicationException("Specified file does not exist: " + softwareHiveFile);
        }

        // pick a random subkey so it doesn't already exist
        var keyPathToCreate = "Microsoft\\Windows\\CurrentVersion\\RunOnceEx\\" + Guid.NewGuid();
        var hKey = RegistryNativeMethods.RegLoadAppKey(softwareHiveFile);
        using (var safeRegistryHandle = new SafeRegistryHandle(new IntPtr(hKey), true))
        using (var appKey = RegistryKey.FromHandle(safeRegistryHandle))
        using (var runOnceExKey = appKey.CreateSubKey(keyPathToCreate))
        {
            runOnceExKey.SetValue("foo", "bar");
            Console.WriteLine("Was able to create {0} and write a value under it", keyPathToCreate);
        }
    }
}

internal static class RegistryNativeMethods
{
    [Flags]
    public enum RegSAM
    {
        AllAccess = 0x000f003f
    }

    private const int REG_PROCESS_APPKEY = 0x00000001;

    // approximated from pinvoke.net's RegLoadKey and RegOpenKey
    // NOTE: changed return from long to int so we could do Win32Exception on it
    [DllImport("advapi32.dll", SetLastError = true)]
    private static extern int RegLoadAppKey(String hiveFile, out int hKey, RegSAM samDesired, int options, int reserved);

    public static int RegLoadAppKey(String hiveFile)
    {
        int hKey;
        int rc = RegLoadAppKey(hiveFile, out hKey, RegSAM.AllAccess, REG_PROCESS_APPKEY, 0);

        if (rc != 0)
        {
            throw new Win32Exception(rc, "Failed during RegLoadAppKey of file " + hiveFile);
        }

        return hKey;
    }
}