We assessed the roles of insulin receptor substrate-1 (IRS-1) and Shc in insulin action on farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) using Chinese hamster ovary (CHO) cells that overexpress wild-type human insulin receptors (CHO-hIR-WT) or mutant insulin receptors lacking the NPEY domain (CHO-DeltaNPEY) or 3T3-L1 fibroblasts transfected with adenoviruses that express the PTB or SAIN domain of IRS-1 and Shc, the pleckstrin homology (PH) domain of IRS-1, or the Src homology 2 (SH2) domain of Shc. Insulin promoted phosphorylation of the alpha-subunit of FTase and GGTase I in CHO-hIR-WT cells, but was without effect in CHO-DeltaNPEY cells. Insulin increased FTase and GGTase I activities and the amounts of prenylated Ras and RhoA proteins in CHO-hIR-WT (but not CHO-DeltaNPEY) cells. Overexpression of the PTB or SAIN domain of IRS-1 (which blocked both IRS-1 and Shc signaling) prevented insulin-stimulated phosphorylation of the FTase and GGTase I alpha-subunit activation of FTase and GGTase I and subsequent increases in prenylated Ras and RhoA proteins. In contrast, overexpression of the IRS-1 PH domain, which impairs IRS-1 (but not Shc) signaling, did not alter insulin action on the prenyltransferases, but completely inhibited the insulin effect on the phosphorylation of IRS-1 and on the activation of phosphatidylinositol 3-kinase and Akt. Finally, overexpression of the Shc SH2 domain completely blocked the insulin effect on FTase and GGTase I activities without interfering with insulin signaling to MAPK. These data suggest that insulin signaling from its receptor to the prenyltransferases FTase and GGTase I is mediated by the Shc pathway, but not the IRS-1/phosphatidylinositol 3-kinase pathway. Shc-mediated insulin signaling to MAPK may be necessary (but not sufficient) for activation of prenyltransferase activity. An additional pathway involving the Shc SH2 domain may be necessary to mediate the insulin effect on FTase and GGTase I.