Objectives: Estrogens are known to modulate lower urinary tract (LUT) trophicity as well as neuronal NO synthase (nNOS) expression in several organs. The aim of this study was to explore the impact of endogenous and of supraestrus levels of estradiol (E2) on LUT and urethral nNOS expression and function. Material and methods: LUT function and histology and urethral nNOS expression were studied in adult female mice subjected either to sham surgery, surgical castration or castration plus chronic E2 supplementation (80 µg.kg-1.day-1, i.e. pregnancy level). Results: The micturition pattern was profoundly altered by long-term supraestrus levels of E2 with decreased frequency paralleled by increased residual volumes higher as compared to ovariectomized mice. Urethral resistance was increased two-fold in E2-treated mice, with no structural changes in urethra supporting a pure tonic mechanism. Acute nNOS inhibition by 7-nitroindazole decreased frequency and increased residual volumes in ovariectomized mice but had no additive effect on the micturition pattern of long-term supraestrus mice, showing that long-term supraestrus E2 levels and acute inhibition of nNOS activity had similar functional effects. Finally, E2 decreased urethral nNOS expression in ovariectomized mice. Conclusion: Long-term supraestrus levels of E2 increased urethral tone through inhibition of nNOS expression whereas physiological levels of E2 had no effect.