Aims: We investigated the roles of neuronal-derived nitric oxide (NO) in the modulation of spontaneous activity of mouse detrusor smooth muscle. Methods: Detrusor smooth muscle strips were isolated from nNOS gene knock-out (nNOS−/−) mice and their wild type siblings (nNOS+/+). The properties of smooth muscle cells were assessed using intracellular electrophysiology and Ca2+ imaging by laser-scanning confocal microscopy. The effects of an nNOS inhibitor, 7-nitro indazole (7-NI) on electrically evoked contractility were assessed using nNOS+/+ mouse detrusor strips. Results: In spontaneously active cells, the frequency of spontaneous action potentials (sAPs) and whole cell Ca2+ flashes in nNOS−/− preparations was lower than that in the nNOS+/+ preparations. The frequency of sAPs was enhanced by a nitric oxide donor, diethylamine NONOate sodium salt (NONOate; 100 µM), both when used alone and when the cGMP pathway was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 10 µM). 7-NI (100 µM) significantly suppressed the electrically evoked contraction of mouse detrusor strips. Conclusions: We suggest that neuronal-derived NO facilitates the generation of spontaneous activity via a cGMP-independent pathway, and consequently enhances the evoked contraction of detrusor. Dysregulation of nNOS containing nerves may underlie bladder pathologies.