In contrast to the 52-kDa Shc isoform, insulin stimulation caused a quantitative, time-dependent decrease in the SDS-PAGE mobility of 66-kDa Shc in both Chinese hamster ovary/IR cells and 3T3L1 adipocytes. Alkaline phosphatase treatment and direct phosphoamino acid analysis demonstrated that insulin stimulated an increase in serine phosphorylation of the 66-kDa isoform but not 52-kDa Shc, although the latter displayed a marked increase in tyrosine phosphorylation. To identify the responsible kinase pathway, we compared the effects on 66-kDa Shc serine phosphorylation by insulin, anisomycin, and osmotic shock, agents that specifically activate the ERK, JNK, or both pathways, respectively. Insulin and osmotic shock both stimulated a decrease in 66-kDa Shc mobility, whereas anisomycin had no effect. Furthermore, expression of a dominant-interfering Ras mutant (N17Ras) prevented the insulin-stimulated, but not the osmotic shock-induced serine phosphorylation of 66-kDa Shc. Consistent with a MEK-dependent pathway mediating 66-kDa Shc serine phosphorylation, the specific MEK inhibitor (PD98059) and expression of a dominant-interfering MEK mutant partially inhibited both the insulin and osmotic shock-induced reduction in 66-kDa Shc mobility. In contrast, expression of the MAP kinase phosphatase (MKP-1) completely prevented ERK activation but did not inhibit the serine phosphorylation of 66-kDa Shc. These data demonstrate that insulin stimulates the serine phosphorylation of the 66-kDa Shc isoform through a MEK-dependent mechanism.