She proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of She in insulin- and EGF-induced activation of p21 ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21 ras . Analysis of She immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in She tyrosine phosphorylation and (ii) She antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of She and the presence of Grb2 and mSOS in She immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, She could be detected as well. Furthermore, She immune complexes contained guanine nucleotide exchange activity toward p21 ras in vitro. From these results, we conclude that after insulin and EGF treatment, She associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21 ras In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with She was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-mSOS with She may be an important event in insulin-induced, mSOS-mediated activation of p21 ras .