Pregnancy is known to induce insulin resistance, but the exact molecular mechanism involved is unknown. In the present study, we have examined the levels and phosphorylation state of the insulin receptor and of insulin receptor substrate 1(IRS-1), as well as the association between IRS-1 and phosphatidylinositol 3-kinase (PI 3-kinase) in the liver and muscle of pregnant rats (day 20 of gestation) by immunoprecipitation and immunoblotting with anti-insulin receptor, anti-IRS-1, anti-PI 3-kinase and antiphosphotyrosine antibodies. There were no changes in the insulin receptor concentration in the liver and muscle of pregnant rats. However, insulin stimulation of receptor autophosphorylation, as determined by immunoblotting with antiphosphotyrosine antibody, was reduced by 30+/-6% (p < 0.02) in muscle and 36+/-5% (p < 0.01) in liver at day 20 of gestation. IRS-1 protein levels decreased by 45+/-6% (p < 0.002) in liver and by 56+/-9% (p < 0.002) in muscle of pregnant rats. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, the insulin-stimulated IRS-1 phosphorylation levels in the muscle and liver of pregnant rats decreased by 70+/-9% (p < 0.01) and 75+/-8% (p < 0.01), respectively. The insulin-stimulated IRS-1 association with PI 3-kinase decreased by 81+/-6% in muscle (p < 0.01) and 79+/-11% (p < 0.01) in the liver during pregnancy. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in pregnancy.402179186