We report here that the product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl reaches a maximum within 1–2 min after stimulation by insulin and gradually declines thereafter. The tyrosine phosphorylation of c-Cbl was also observed after treatment of 3T3-L1 adipocytes with epidermal growth factor, whereas platelet-derived growth factor had no effect. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with fusion proteins containing the Src homology 2 (SH2) domains of Crk and the Fyn tyrosine kinase, but not with fusion proteins containing the SH2 domains of either the p85 subunit of phosphatidylinositol 3′-kinase or the tyrosine phosphatase SHPTP2/Syp. Furthermore insulin stimulates the association of c-Cbl with endogenous c-Crk and Fyn in intact 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl is regulated during adipocyte differentiation. Neither insulin-like growth factor 1 nor insulin stimulated the tyrosine phosphorylation of c-Cbl in 3T3-L1 fibroblasts. Moreover, c-Cbl is not tyrosine phosphorylated in response to insulin in cells expressing high levels of the human insulin receptor, or in hepatocytes, despite comparable levels of c-Cbl expression. These results suggest that c-Cbl might have a novel function in the regulation of insulin receptor intracellular signalling in 3T3-L1 adipocytes.