The purpose of this study was to investigate the effect of the endogenous cannabinoid anandamide on the nonadrenergic noncholinergic (NANC) relaxant responses to electrical field stimulation in isolated rat corpus cavernosum. The corporal strips were mounted under tension in a standard oxygenated organ bath with guanethidine sulfate (5 μM) and atropine (1 μM) (to produce adrenergic and cholinergic blockade). The strips were precontracted with phenylephrine hydrochloride (7.5 μM) and electrical field stimulation was applied at different frequencies to obtain NANC-mediated relaxation. The expression of CB1, CB2 and vanilloid receptor proteins within the rat corpus cavernosum was evaluated using western blot analysis. The results showed that the relaxant responses to electrical stimulation were significantly enhanced in the presence of anandamide at 1 and 3 μM. The potentiating effect of anandamide (1 μM) on relaxation responses was significantly attenuated by either the selective cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; 1 μM) or the vanilloid receptor antagonist capsazepine (3 μM), but not by the selective cannabinoid CB2 receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl) ethyl]-1H-indol-3-yl (4-methoxyphenyl)methanone (AM630; 1 μM). Neither of these antagonists had influence on relaxation responses. Indomethacin (20 μM) had no effect on NANC-mediated relaxation in the presence or absence of anandamide (1 μM). Preincubation with Nw-Nitro-l-Arginine Methyl Ester (l-NAME; 1 μM) significantly inhibited the relaxation responses in the presence or absence of 1 μM anandamide. Although at 30 nM, l-NAME did not cause a significant inhibition of relaxant responses individually, it significantly inhibited the potentiating effect of anandamide (1 μM) on relaxation responses. Anandamide (1 μM) had no influence on concentration-dependent relaxant responses to sodium nitroprusside (10 nM–1 mM), a nitric oxide (NO) donor. The western blotting of corporal tissues demonstrated the existence of both vanilloid and CB1 receptors in corporal strips. In conclusion, our results showed that anandamide has a potentiating effect on NANC-mediated relaxation of rat corpus cavernosum through both CB1 and vanilloid receptors and the NO-mediated component of the NANC relaxant responses to electrical stimulation is involved in this enhancement.