The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by G-nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and G-nucleotide dissociation inhibitors (GDIs). Little is known how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert RacGTP to RacGDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to RacGTP and catalyzes the conversion of bound GTP to GDP on Rac. The presence of activated Rac diminished the RhoGDIalpha-Bcr interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of the RacGTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, which indicates that binding of RhoGDIalpha and RacGTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates the Bcr GAP activity.