TFIID is a multisubunit protein complex comprised of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). The TAFs in TFIID are essential for activator-dependent transcription, The cloning of a complementary DNA encoding a human TFIID TAF, TAF(II)55, that has no known homolog in Drosophila TFIID is now described. TAF(II)55 is shown to interact with the largest subunit(TAF(II)230) of human TFIID through its central region and with multiple activators-including Sp1, YY1, USF, CTF, adenoviral E1A, and human immunodeficiency virus-type 1 Tat proteins-through a distinct amino-terminal domain. The TAF(II)55-interacting region of Sp1 was localized to its DNA-binding domain, which is distinct from the glutamine-rich activation domains previously shown to interact with Drosophila TAF(II)110. Thus, this human TFIID TAF may be a co-activator that mediates a response to multiple activators through a distinct mechanism.