Insulin-like growth factor I (IGF-I) stimulates mitogenesis in proliferating 3T3-L1 preadipocytes. However, IGF-I functions to stimulate differentiation once growth arrest occurs at confluence. Epidermal growth factor (EGF) is also a potent mitogen in these cells, but inhibits differentiation of preadipocytes. We compared mitogenic signaling via the mitogen-activated protein kinase (MAPK) pathway in response to IGF-I or EGF in proliferating, growth-arrested, and differentiating 3T3-L1 cells. IGF-I stimulation of MAPK was rapid and maximal in proliferating 3T3-L1 preadipocytes, but decreased greatly in differentiating cells. EGF was more potent than IGF-I in stimulating MAPK activity in 3T3-L1 cells, and activation of MAPK was maintained in differentiating cells. These results suggest an uncoupling of MAPK activation specific to IGF-I-mediated 3T3-L1 preadipocyte differentiation. Studies of proximal signaling revealed Shc phosphorylation and Shc/Grb2 complex formation in IGF-I-treated proliferating, but not differentiating, cells. Insulin receptor substrate-1 phosphorylation after IGF-I treatment was present in proliferating, quiescent, and differentiating preadipocytes. Shc phosphorylation and Grb2 association after EGF treatment were present throughout all phases of growth. The change in IGF-I signaling via Shc phosphorylation and MAPK activity during 3T3-L1 differentiation indicates that loss of IGF-I mitogenic signaling via the MAPK pathway is part of the differentiation process.