A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting with CIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1.