The uterine vasculature plays an important role during pregnancy, by providing adequate perfusion of the maternal-fetal interface. To this end, substantial remodelling of the uterine vasculature occurs with consequent changes in the responsiveness to contractile agents. The purpose of our study was to characterize the vasorelaxant effects of estrogens on vascular smooth muscles of the rat uterine artery during pregnancy and to evaluate the involvement of estrogen receptors (ESR) and nitric oxide synthases (NOS). To do so, NOS expression in the whole uterine and mesenteric circulatory bed was measured by Western blotting. The vasorelaxant effects of 17-estradiol (17-E₂) were assessed on endothelium-denuded uterine arteries using wire myographs in the absence and presence of pharmacological modulators (L-NAME, ICI 182,780, tamoxifen). All experiments were performed on arteries from non-pregnant (NP) and late pregnant (P) rats. In the uterine vasculature of the latter group, NOS3 (eNOS) expression was increased, while NOS1 (nNOS) was reduced compared to NP rats. Expression of the NOS2 (iNOS) isoform was undetectable in the two groups. Both 17-E₂ and 17-E₂ induced the uterine artery relaxation, but the latter evoked lower responses. Endothelium-denuded arteries from NP rats showed larger relaxation with 17-E₂ than P rats. This larger relaxation disappeared in the presence of L-NAME. The ESR antagonist ICI 182,780 did not affect acute relaxation with 17β-E₂ and 17-E₂. Moreover, membrane-nonpermeant 17-E₂:BSA (estradiol conjugated to bovine serum albumin) did not induce any vasorelaxation. Our results indicate that estrogens exert direct acute vasorelaxant effects in smooth muscles of the rat uterine artery that are mediated by mechanisms independent of ESR activation, but with some stereospecificity. Part of this effect, in NP rats only, is due to nitric ocide produced from muscle NOS1.