http://www.vistax64.com/vista-general/72880-win16-application-support.html
Will I be able to run Win16 (Windows 3.1) applications in Vista?
Many DOS and 16-bit Windows applications run fine
on 32-bit Vista (any edition).
My understanding - based on answers during the beta - is that backwards
compatibility remains a significant goal for the Windows team. The NTVDM and
16 bit WoW are fully functional in Vista; in fact they've been enhanced
beyond what was available in XP (corrections to interrupt vector table,
etc).