Could you try the sample Clickonce applications up on -http://clickonce.members.winisp.net/
There are no known issues with ClickOnce on WIndows 2000. A successful installation of the framework redist should give you all the client settings needed to run a Click once application.
Does it repro for all Win2k machines? If not, how easy is it to get into this state? Was the VS 2005 installation successful ont hese machiens or were there soem failures?