We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.