Estrogens exert multiple genomic effects on adipose tissue through binding to nuclear estrogen receptors. However, there are several evidences for additional nongenomic mechanisms whereby estrogens may exert their control on adipose tissue metabolism through rapid activation of various membrane-initiated kinase cascades. Here, we tested rapid effects of estrogens on NO production in white adipose tissue using 17- estradiol (E2) and its membrane impermeant albumine conjugated form (E2-BSA). We found that both E2 and E2-BSA stimulate NOS activity in adipocytes. These effects were abolished by 1) ICI 182-780, a selective estrogen receptor antagonist; 2) wortmannin, an inhibitor of phosphatidylinositol 3-kinase and 3) N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89) an inhibitor of protein kinase A. In contrast to NOS activation by E2_, E2-BSA-induced NOS activity was abolished by UO126, an inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (p42/p44 MAP kinases). Immunoblotting studies have shown that both estrogens phosphorylate NOS III on Ser¹¹⁷⁹, an effect which is prevented by wortmannin and H89, suggesting that NOS III is the target for estrogen-induced NOS activity. Furthermore only the E2-BSA-induced NOS III phosphorylation on Ser¹¹⁷⁹ was totally abolished by UO126. These results indicate that the signaling cascades involved in adipocyte NOS stimulation by estrogens are different depending on whether estrogens are free or conjugated to albumin, and therefore underline the importance of ER locations in the nongenomic actions of estrogens in these cells.