The effects of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) were determined on cholinergic contractility in strips of human colonic longitudinal muscle and circular muscle in vitro, in the presence of nitric oxide synthase blockade with N-nitro-l-arginine (10(-4) M). Anandamide and 2-AG inhibited longitudinal muscle and circular muscle contractile responses to acetylcholine (10(-9)-10(-4) M) in a concentration-dependent manner. This was unaltered following pretreatment with the cannabinoid CB(1) receptor-selective antagonist AM251 (10(-7) M), however in isolation AM251 elicited a significant rightward shift in the potency of acetylcholine-evoked contraction in both longitudinal muscle and circular muscle preparations. Pretreatment with an inhibitor of anandamide catabolism, arachidonoyl trifluoromethyl ketone (10(-5) M), alone caused a significant decrease in the potency of acetylcholine-evoked contraction in both longitudinal and circular muscle, but had no significant additional effect on the anandamide-induced (10(-5) M) suppression of contraction. Pretreatment with the cannabinoid CB(2) receptor inverse agonist JTE 907 (10(-6) M) neither influenced the potency of acetylcholine-evoked contraction alone nor prevented the potency shift in acetylcholine-evoked contraction in the presence of anandamide (10(-5) M). The findings of the present study indicate that the endocannabinoids anandamide and 2-arachidonoylglycerol suppress colonic cholinergic contractility via a non conventional cannabinoid or non-cannabinoid receptor-mediated pathway. Cholinergic contraction may be tonically modulated by endocannabinoids and/or products of arachidonate metabolism unrelated to endocannabinoid production. The extent of anandamide metabolism is not sufficient to influence the functional effects of its exogenous administration in human colonic tissue in vitro.