Only exception is NT/2000 (3.5 doesn't install easily in most cases)
In which case , Your best bet is testing .the prerequisites may not install on 2000 and NT
(this has been documented by Microsoft)
In order to properly install .net framework 3.5 on the older OS it requires KB 835732 , Dll's loaded in proper folder and etc. etc.
If your overall question is "I'm writing a spam program and I want to know if I can mark the working OS' as NT 4.0 , 2000/ XP / Vista / 7 , I say leave out NT and 2000 as compatible OS.
just a IMO thing