I have also done a similar action and later removed the second HDD without any Problem.
No Bootup errors afterwards. So it is not a "bug" or something related to the OS.
Tell us how the Disks are connected ( S-Ata / IDE ) to the Motherboard.
Have you rejumpered the Disks ( Master / Slave ) , e.g. is the now disconnected HDD still present in BIOS ?
How have you used the Disk in Vista ?
Did you made shortcuts in Vista that point to files residing on the Second ( now disconnected ) HDD so that vista cannot find the Originals anymore?
Have you tried to boot the PC from the Vista-DVD and used the "fix boot-problems" option ?
( Item under "install now" )
Please explain a bit more what exactly you have done before you removed the Disk or if problem is solved.
SBJ