TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAFIIs). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAFIIs. Biochemical studies suggested that the Drosophila (d) TFIID complexes contain only eight TAFIIs, leaving a number of yeast and human TAFIIs (e.g., hTAFII55, hTAFII30, and hTAFII18) without known Drosophila homologues. We demonstrate that Drosophila has not one but two hTAFII30 homologues, dTAFII16 and dTAFII24, which are encoded by two adjacent genes. These two genes are localized in a head-to-head orientation, and their 5′ extremities overlap. We show that these novel dTAFIIs are expressed and that they are both associated with TBP and other bona fide dTAFIIs in dTFIID complexes. dTAFII24, but not dTAFII16, was also found to be associated with the histone acetyltransferase (HAT) dGCN5. Thus, dTAFII16 and dTAFII24 are functional homologues of hTAFII30, and this is the first demonstration that a TAFII-GCN5-HAT complex exists in Drosophila. The two dTAFIIs are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells. These results together indicate that dTAFII16 and dTAFII24 may have similar but not identical functions.