Recent in vivo and in vitro studies suggest that nitric oxide or a nitric-oxide-like substance mediates nonadrenergic, noncholinergic relaxation of trabecular smooth muscle through activation of the cyclic guanosine monophosphate (cGMP) pathway. In 60 Sprague-Dawley rats, we investigated the effect of intracavernous administration of different drugs known to act at different levels of the cyclic adenosine monophosphate (cAMP) and cGMP pathways. Neither cAMP nor drugs that stimulate adenylate cyclase activity (vasoactive intestinal peptide, prostaglandin E(1), calcitonin gene-related peptide) provoked any change in the basal intracavernous pressure. N-ethylmaleimide, an inhibitor of the enzyme adenylate cyclase, did not modify the response to electrostimulation of the cavernous nerve, indicating that the cAMP pathway does not play a significant role in penile erection in rats. However, intracavernous administration of methylene blue, a guanylate cyclase inhibitor, significantly reduced the response to electrostimulation (p = 0.001). Direct intracavernous injection of cGMP caused a statistically significant, dose-dependent increase in intravemous pressure that was not significantly inhibited by methylene blue. Sodium nitroprusside, a nitric oxide releaser and therefore a guanylate cyclase activator, caused a dose-dependent increase in intracavernous pressure (p < 0.05) that was inhibited almost completely by methylene blue (p = 0.002), supporting the theory that nitric oxide activates the synthesis of cGMP and that cGMP causes cavernous smooth muscle relaxation. Papaverine elicited an intracavernous pressure increase that was not affected by methylene blue or N-ethylmaleimide, indicating that papaverine acts through an independent pathway. N-methyl-D-aspartate and L-glutamic acid, excitatory amino acids that act in the central nervous system by stimulating nitric oxide synthase, had no effect on the intracavernous pressure. 5-Hydroxytryptamine, which is believed to cause presynaptic inhibition of release of excitatory amino acids, caused a potent inhibition of the cavernous response to electrostimulation (p < 0.001). It appears that cavernous smooth muscle relaxation is mediated by the release of a nitric-oxide-like substance with subsequent activation of guanylate cyclase. The cAMP system seems to play no important role in this mechanism in the rat.