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There are several things you could try. I’ll just copy paste my solution from the AG forum =) 

MS-DOS or 16-bit Windows-based program Error Message

This installation program will reinstall the missing or corrupt Windows XP system files, autoexec.nt and config.nt. The absence or corruption of one or more of these files causes a “16 Bit Subsystem” error.

XP_Fix.EXE by TRF Systems, Inc. 

Problems from other users:

Running Audit on my computer showed that a program called WINDUPDATE.EXE (not WINUPDATE.EXE) was deleting my Autoexec.nt file on boot up. If I replaced the file it would delete it again.
Go to \Program Files\WINDUPDATE folder and delete it!

Go to your registry and delete anything named WINDUPDATE. Put the Autoexec.nt file back into your \system32 folder and all is well! This was definately the
problem here and I bet most everyone has the same nasty bugger, WINDUPDATE.EXE on their infected computer.

Another note and workaround:

C:\Windows\System32\Autoexec.nt The system file is not suitable for running MS-DOS and Microsoft Windows applications. Choose ‘Close’ to terminate the application.

This problem just started around mid August 2004, so I suspect it to be related to a recent Microsoft Update that you may have downloaded for Windows XP.

HomeSchool Easy Records (the current version) is a Windows program (not a DOS program) but it is a “16 bit” Windows program. Apparently HER and other 16 bit Windows programs will run into this error when you try to run them. Note: HER version 3, now in the works, is a 32 bit Windows program, so it won’t be prone to this problem.

There is a cure. Please read the following tip and follow the steps to reinstall three files: autoexec.nt, config.nt, and

Side note: You also want to look for the file “windupdate.exe” (note the spelling closely — the ‘d’) and if you find it anywhere on your hard drive, delete it! This is a secondary problem and you are not very likely to find this file. But if you have it, it actually deletes the autoexec.nt file each time you start your PC, so even if you restore the file, it will get deleted again!

Here is the tip originally posted to the Internet by Spencer Greystrong:

This article…kb;en-us;324767  is the Microsoft response to that error message. The fix involves expanding the original files from your XP CD and overwriting. The problem with that solution is that many people do not have the original CD. [So the links below were created so you can get the files directly].

When you click on either link below you will see a file-download window appear. Click on the “Open” button and you will get a WinZip self-extractor window. Leave the “Unzip to folder” set to “cwindows\system32” and leave the “Overwrite files without prompting” check box checked. Ignore the yellow warning sign (this is trustworthy code). Click on the “Unzip” button. The files will save to your hard drive. Then click on the “Close” button.

For Windows XP Professional click on this link:…/XPProfiles.exe 
windupdate.exe is spyware and will read as: DPF: {15AD4789-CDB4-47E1-A9DA-992EE8E6BAD6} –…0f91b20c15a3e0e 

Hope this helps