There are many clinical and experimental reports demonstrating that estrogens and insulin interact when affecting their target organs. Estrogen receptors consist of two isoforms, estrogen receptors-alpha (ER-α) and -beta (ER-β), but their roles in insulin-induced glucose uptake in mature adipose tissue have yet to be clarified. To evaluate the roles of ER-α, expressed predominantly in adipocytes, we have investigated the effects of estradiol (E2), an ER-α selective agonist (PPT), and its selective antagonist (MPP) on glucose uptake and insulin action in 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed to E2 or PPT and/or MPP at different concentrations. The cells were then subjected to 2-deoxy-D-glucose transport assay, western blot analysis, or RT-PCR analysis. Treatment of these cells with E2 or PPT resulted in biphasic effects on glucose transport, that is high (10^-5 M or 3 × 10^-6 M each) and low (10^-8 M) doses produced inhibition and stimulation, respectively. The favorable effect observed at 10^-8 M of E2 was diminished by treatment with MPP. Western bolt analysis revealed that these effects of E2, PPT and MPP paralleled the level of IRS-1 tyrosine phosphorylation. However, IRS-1 serine phosphorylation, suppressor of cytokine signaling (SOCS)-1,-2,-3 and protein tyrosine phosphatase 1B (PTP1B) expression did not change compaired to control subjects. Our data clearly show that ER-α contributes to insulin stimulated glucose uptake through regulation of the tyrosine phosphorylation of IRS-1 protein.