Nitric oxide (NO), a neurotransmitter in the lower urinary tract, stimulates soluble guanylyl cyclase (sGC) and in turn cGMP-dependent protein kinase G (PKG) to modulate a number of downstream targets. NO donors reduce bladder hyperactivity in some pathological models, but do not affect normal bladder activity in the adult rat. In this study, the NO donor SNAP (100 µM) decreased the amplitude and frequency of spontaneous and of carbachol-enhanced contractions in neonatal rat bladder strips, which are intrinsically hyperactive. This effect was blocked by inhibition of sGC and mimicked by application of a membrane permeable cGMP analogue (8-bromo-cGMP, 100 µM). Inhibition of PKG prevented or reversed the inhibitory effects of 8-bromo-cGMP. A portion of the SNAP-mediated inhibition was also dependent upon PKG; however, a short lasting, sGC-dependent inhibitory effect of SNAP was still present after PKG inhibition. Inhibition of NO synthase with L-NAME (100 µM) did not change the amplitude or frequency of contractions. However, inhibition of endogenous phosphodiesterase (PDE)-5 with zaprinast (25 µM) reduced the amplitude and frequency of phasic contractions and increased the magnitude of inhibition produced by maximal concentrations of SNAP, suggesting that endogenous PDEs are constitutively active and regulate cGMP production. These results suggest that the NO-cGMP-PKG pathway may be involved in inhibitory control of the neonatal rat bladder.