Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate enzymes required for cholesterol and fatty acid synthesis. Expression of SREBP-1 is enhanced by insulin; however, the actual insulin-signaling cascades employed are yet unclear. We determined the roles of the phosphatidylinositol (PI) 3-kinase and mitogen-activated protein (MAP) kinase-dependent pathways in the effect of mediating insulin on SREBP-1 in L-6 skeletal muscle cells and 3T3 L1 adipocytes, using wortmannin or LY294002 to inhibit the PI 3-kinase pathway, and PD98059 to inhibit the MAP kinase-dependent pathway. In myocytes, insulin increased SREBP-1 protein in a dose-dependent manner. 1 and 10 nm insulin significantly increased expression of total cellular SREBP-1 protein at 24 and 48 h, nuclear SREBP-1 protein at 24 h, and SREBP-1a mRNA at 24 h. Although wortmannin and LY294002 had no effect on this aspect of insulin action, PD98059 completely blocked each of these responses. Transfection of a dominant negative mutant of Ras similarly blocked the insulin effect on SREBP-1. In contrast, in adipocytes, the insulin effect on SREBP-1 was mediated via the PI 3-kinase and not the MAP kinase pathway. In conclusion, although insulin increases skeletal muscle SREBP-1 expression in a dose-dependent fashion via the MAP kinase-dependent signaling pathway, insulin action on adipocyte SREBP-1 is mediated via the PI 3-kinase signaling pathway. In the state of insulin resistance, characterized by selective inhibition of the PI 3-kinase pathway, the usual stimulation of lipogenesis by insulin in adipocytes may be inhibited, whereas intramyocellular lipogenesis via the MAP kinase pathway of insulin may continue unabated.