Phospholipase C-gamma (PLCgamma) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. In this paper, we present evidence for the association of the insulin receptor (IR) with PLCgamma. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLCgamma and coimmunoprecipitation of the IR and PLCgamma were observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLCgamma and the IR, we used a specific inhibitor of PLC, U73122, or microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLCgamma to block insulin-stimulated GLUT4 translocation. We demonstrate inhibition of 2-deoxyglucose uptake in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. We conclude that PLCgamma is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes.