To elucidate the neuropharmacology of erection, we undertook an in vivo canine study to examine the role of cholinergic and nonadrenergic, noncholinergic (NANC) neuroeffectors and the sinusoidal endothelium in erection induced by electrostimulation. We also examined the effect of adenylate cyclase and guanylate cyclase blockers by intravenous injection of N-ethylmaleimide and methylene blue, respectively. In addition, the effects of intracavernous injection of the nitric oxide-releasing substance, nitroprusside, and bromocyclic adenosine monophosphate (AMP) and bromocyclic guanosine monophosphate (GMP) were also studied. In contrast to in vitro results, atropine reduced the increase of intracavernous pressure after neurostimulation (p = 0.029). Intracavernous injection of CHAPS to destroy the sinusoidal endothelium abolished the response to acetylcholine (p = 0.001), but only partially inhibited the response to electrostimulation (mean = 75% pressure increase, p = 0.022), indicating that neuronal nitric oxide plays a major role in penile erection. Methylene blue, a guanylate cyclase inhibitor, significantly inhibited the erectile response to both neurostimulation and sodium nitroprusside (p = 0.000 and 0.017, respectively). However, N-ethylmaleimide, an adenylate cyclase inhibitor, could not reduce the response to neurostimulation (p = 0.078). The erectile response to intracavernous injection of cGMP was significantly better than that induced by cAMP (p = 0.025). Our results suggest that both the cholinergic and NANC neuroeffectors and the sinusoidal endothelium are involved in erection. In addition, our data imply that the neuronal nitric oxide/cyclic GMP system is the most likely pathway for penile smooth muscle relaxation and erection.