Insulin activates hexose transport via at least two mechanisms: a p21ras-dependent pathway, leading to an increase in the amount of cell surface GLUT1; and a metabolic, p21ras-independent pathway, leading to translocation of the insulin-responsive transporter GLUT4 to the cell surface. Following insulin stimulation, SHPTP2, a non-transmembrane protein-tyrosine phosphatase, associates with insulin receptor substrate 1 via its Src homology 2 (SH2) domains. Microinjection of a glutathione S-transferase fusion protein encoding the N- and C-terminal SH2 domains of SHPTP2 (GST-NC-SH2) or anti-SHPTP2 antibodies into NIH-3T3 fibroblasts overexpressing the insulin receptor blocks insulin-induced DNA synthesis. Microinjection of either GST-NC-SH2 or anti-SHPTP2 antibodies into 3T3-L1 adipocytes inhibited the insulin-stimulated increase in expression of GLUT1. In contrast, translocation of GLUT4 to the cell surface was unaffected by either GST-NC-SH2 or anti-SHPTP2 antibodies. These data confirm a role for SHPTP2 in insulin-stimulated mitogenesis and indicate that whereas SHPTP2 is necessary for insulin-stimulated expression of GLUT1, it is not required for activation of the metabolic pathway leading to GLUT4 translocation.