The small GTP-binding protein Cdc42, the guanine nucleotide exchange factor Scd1, the p21-activated kinase Shk1, and the adaptor protein Scd2 are involved in the Cdc42-dependent signaling cascade in fission yeast. In the present study, we analyzed the Cdc42 binding and scaffolding activities of Scd2 by co-precipitation assays. We found that two SH3-containing regions, amino acid residues 1-87 (CB1 (Cdc42-binding region 1)) and 110-266 (CB2), of Scd2 can bind to the GTP-bound form of Cdc42. CB2 is cryptic because of the intramolecular binding between the SH3 domain in CB2 (SH3(C)) and the PX domain and binds to Cdc42 only when the Scd2 PB1 domain binds to the PC motif-containing region (residues 760-872) of Scd1. This CB2.Cdc42 association, which would stabilize the open configuration of Scd2, enables the SH3(C) domain to bind to the polyproline motif of Shk1. We also found that the GTP-bound form of Cdc42 binds to the CRIB motif of Shk1 more strongly than to Scd2. Thus, Scd2 functions as a scaffold to form a protein complex, and the GTP-bound Cdc42 might be transferred effectively from the upstream activator Scd1 to the downstream effector Shk1 via Scd2.