The effects of estrogen (E₂), progesterone (P₄), and E₂ and P₄ (E₂ P₄) on uterine, vaginal, and cerebellar nitric oxide synthase (NOS) were examined. Additionally, experiments were done to investigate whether NOS-containing nerves were present in the uterus and vagina and the extent to which vaginal smooth muscle response was dependent on nitric oxide (NO). Cytosolic NOS was determined by the formation of [¹⁴C]citrulline from [¹⁴C]arginine, and NOS localization was visualized by immunohistochemistry. Vaginal smooth muscle relaxation was induced by electrical field stimulations (EFS). NOS activity in the uterus was markedly down-regulated in all hormone-treated groups. Vaginal NOS activity was nearly 4-fold higher than the uterine NOS activity and was considerably reduced by E₂ or E₂ P₄ treatment. In contrast to findings in the uterus, P₄ treatment up-regulated vaginal NOS. Hormone treatment had no significant effect on cerebellar NOS. NOS-containing nerves could be demonstrated in the uterus and vagina by immunohistochemistry. Vaginal smooth muscle responded with relaxation after EFS, which was inhibited by N^G-nitro-l-arginine. A relatively high vaginal NOS, a down-regulation by E₂, an up-regulation by P₄, and NO-dependent response of vaginal smooth muscle suggest a tissue-specific physiological role.