The TATA box binding protein (TBP) plays a central and essential role in transcription initiation. At TATA box-containing genes transcribed by RNA polymerase II, TBP binds to the promoter and initiates the assembly of a multiprotein preinitiation complex. Several studies have suggested that binding of TBP to the TATA box is an important regulatory step in transcription initiation in vitro. To determine whether TBP is a target of regulatory factors in vivo, we performed a genetic screen in yeast for TBP mutants defective in activated transcription. One class of TBP mutants identified in this screen comprises inositol auxotrophs that are also defective in using galactose as a carbon source. These phenotypes are due to promoter-specific defects in transcription initiation that are governed by the upstream activating sequence (UAS) and apparently not by the sequence of the TATA element. The finding that these TBP mutants are severely impaired in DNA binding in vitro suggests that transcription initiation at certain genes is regulated at the level of TATA box binding by TBP in vivo.