Pax-5 codes for the transcription factor BSAP, which plays an important role in midbrain patterning, B cell development, and lymphoma formation. Pax-5 is known to control gene expression by recognizing its target genes via the NH2-terminal paired domain and by regulating transcription through a COOH-terminal regulatory module consisting of activating and inhibitory sequences. The central region of Pax-5 contains a sequence with significant homology to the first alpha-helix of the paired-type homeodomain. This partial homeodomain has been highly conserved throughout vertebrate evolution because it is found not only in Pax-5 but also in the related Pax-2 and Pax-8 members of the same Pax subfamily. Here we report that the partial homeodomain binds the TATA-binding protein (TBP) and retinoblastoma (Rb) gene product. Both TBP and Rb were shown by coimmunoprecipitation experiments to directly associate with Pax-5 in vivo. The conserved core domain of TBP and the pocket region as well as COOH-terminal sequences of Rb are required for interaction with the partial homeodomain of Pax-5 in in vitro binding assays. Furthermore, Pax-5 was specifically bound only by the underphosphorylated form of Rb. These data indicate that Pax-5 is able to contact the basal transcription machinery through the TBP-containing initiation factor TFIID, and that its activity can be controlled by the cell cycle-regulated association with Rb.