Recombinant TFIID proteins from yeast, Drosophila, and human function interchangeably in vitro to restore basal level transcription to a human HeLa extract depleted for TFIID. Here we report that the recently cloned human and Drosophila TFIID genes fail to substitute in vivo for the S. cerevisiae TFIID gene, SPT15, which is essential for viability. Analysis of yeast-human hybrid TFIID proteins reveals that the failure of human TFIID to functionally replace yeast TFIID maps to the highly conserved C-terminal domain. Thus, the C-terminal conserved domain of TFIID, as well as the N-terminal divergent domain, appears to be involved in species-specific interactions.