The human immunodeficiency virus-1 (HIV-1) protein Tat, encoded by one of the HIV regulatory genes, tat, is reported to be essential for HIV gene expression and replication in infected cells. Observations suggest that several cellular factors cooperate with Tat in this process. Tat binding protein-1 (TBP-1) is reported to be one such cellular factor that specifically suppresses Tat-mediated transactivation of HIV replication in vitro. Here we have cloned a novel factor, TBP-1 interacting protein (TBPIP) from the mouse, which interacts with mouse TBP-1. TBPIP contains several kinase phosphorylation sites and co-localizes with TBP-1 in vivo. The fact that Tat activity is altered synergistically by the TBP-1 and an additional TBP-1 binding protein has not been reported before. We provide evidence that expression of TBPIP enhances the inhibitory action of TBP-1 on Tat-mediated transactivation in vitro. Our results suggest that TBPIP may have a key role in suppressing the Tat-mediated transactivation.