The endogenous cannabinoid receptor ligand, anandamide, produced a concentration related inhibition of electrically evoked contractions of the guinea-pig myenteric plexus preparation. Its potency was markedly enhanced by phenylmethylsulphonyl fluoride (2.0-200 microM) which presumably acts by inhibiting the hydrolysis of anandamide in this preparation. The degree of this potentiation increased with the concentration of phenylmethylsulphonyl fluoride used. The methyl analogue of anandamide, R-(+)-arachidonyl-1'-hydroxy-2'-propylamide, also inhibited contractions of the guinea-pig myenteric plexus preparation. The potency of this compound was much less affected by phenylmethylsulphonyl fluoride than was the potency of anandamide, confirming its greater resistance to hydrolysis. Phenylmethylsulphonyl fluoride did not alter the inhibitory potency of the cannabinoid, CP 55,940 ((-)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4- [3-hydroxypropyl]cyclohexan-1-ol), which is not an amidase substrate. Nor did phenylmethylsulphonyl fluoride affect the ability of anandamide to inhibit electrically evoked contractions of the mouse vas deferens, suggesting that anandamide does not undergo hydrolysis in this tissue.