The Pas GTPase-activating protein p120(GAP) is a multidomain protein consisting of a variety of noncatalytic domains that may be involved in its regulation. RACK1 is a membrane-associated protein that binds the C2 domain of PKC and is related in sequence to the beta subunit of heterotrimeric G-proteins which has been implicated in binding to PH domains. Because p120(GAP) contains both PH and C2/CaLB domains we determined whether it is also a RACK1 binding protein. Coimmunoprecipitation experiments indicate that p120(GAP) associates with RACK1, whereas PH or C2/CaLB domain deletion mutants do not. A fusion protein containing the GAP PH domain bound to endogenous RACK1 in lysates in a concentration-dependent manner and directly associated with recombinant RACK1. Finally, serine/threonine phosphorylation appears to be involved in regulating this association. These results suggest that p120(GAP) and RACK1 interact in vivo in a manner dependent upon both the PH and C2/CaLB domains of GAP.