The endogenous cannabinoid, anandamide, has been suggested as an endothelium-derived hyperpolarizing factor (EDHF). We found that anandamide-evoked relaxation in isolated segments of rat mesenteric artery was associated with smooth muscle hyperpolarization. However, although anandamide-evoked relaxation was inhibited by either charybdotoxin (ChTX) or iberiotoxin, inhibition of the relaxation to EDHF required a combination of ChTX and apamin. The relaxations induced by either anandamide or EDHF were not inhibited by the cannabinoid receptor (CB1) antagonist SRI41716A, or mimicked by selective CB1 agonists. Thus, anandamide appears to cause smooth muscle relaxation via a CB1 receptor-independent mechanism and cannabinoid receptor activation apparently does not contribute to EDHF-mediated relaxation in this resistance artery.