TFIID, the general transcription factor that binds TATA promoter elements, is highly conserved throughout the eukaryotic kingdom. TFIIDs from different organisms contain C-terminal core domains that are at least 80% identical and display similar biochemical properties. Despite these similarities, yeast cells containing human TFIID instead of the endogenous yeast protein grow extremely poorly. Surprisingly, this functional distinction reflects differences in the core domains, not the divergent N-terminal regions. The N-terminal region is unimportant for the essential function(s) of yeast TFIID because expression of the core domain permits efficient cell growth. Analysis of yeast-human hybrid TFIIDs indicates that several regions within the conserved core account for the phenotypic difference, with some regions being more important than others. This species specificity might reflect differences in DNA-binding properties and/or interactions with activator proteins or other components of the RNA polymerase II transcription machinery.