To observe the effect of testosterone on the frequency of urination in mice lacking neuronal nitric oxide synthase (nNOS(-/-)), we compared the urination patterns between unanaesthetized male wild-type (n = 27) and nNOS(-/-) mice (n = 50) with or without testosterone treatment. Compared with wild-type mice, nNOS(-/-) mice showed a greater frequency of urination during a 24-h observation period (3.0 vs. 5.4 times/day, p < 0.0001) without any significant difference in the total voided volume or the functional voiding capacity. While testosterone treatment did not affect the urination patterns in wild-type, it decreased the daytime frequency of urination (5.4 vs. 3.7 times, p = 0.0198) and the nighttime urination (4.4 vs. 2.9 times, p = 0.039) in nNOS(-/-) mice. The nNOS(-/-) mice can be a useful animal model for urinary frequency. Testosterone improved the functional abnormalities in the voiding of nNOS(-/-) mice.