We report the cloning of the gene that encodes the yeast TATA binding protein TFIID. TFIID contains 240 amino acids and has no obvious sequence similarity to other known proteins. TFIID was synthesized in vitro and in two separate assays behaved identically to the protein purified from yeast. TFIID bound to TATA elements from the adenovirus major late promoter (TATAAAA) and the yeast LEU2 promoter (TATTTAA) and formed protein-DNA complexes stable to electrophoresis only in the presence of TFIIA. In vitro-synthesized yeast TFIID also complemented a mammalian in vitro transcription system that lacked TFIID. Comparison of the yeast TFIID gene with the yeast SPT15 gene (suppressor of Ty element insertions) showed that the two genes are identical. This finding indicates that the yeast TFIID activity defined in vitro is responsible for specific transcription in vivo.