To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.