The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada.Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada.Spt complexes.