Using a combination of ion exchange and immunoaffinity chromatography we have purified the general transcription initiation factor TFIID to near homogeneity from Saccharomyces cerevisiae. Yeast TFIID is composed of TBP, the TATA box binding protein, and 14 distinct TBP-associated factors (TAFs), which range in size from 17 to 150 kDa. Twelve of the TAF subunits have been previously identified, but two, TAF48p and TAF65p, are novel. TAF48p exhibits significant sequence similarity to the conserved C-terminal region of Drosophila TAF110p, human TAF130p, and human TAF105p and is encoded by a previously identified gene MPT1. TAF65p shows no significant sequence homology to any previously identified TAFp. The genes encoding TAF48p and TAF65p are single copy and essential for normal yeast cell growth. Furthermore, neither TAF48p nor TAF65p are associated with the histone acetylase Spt-Ada-Gcn5 complex or other non-TFIID TBF.TAF complexes. The significance of these results in terms of TFIID structure, function, and organization is discussed.