A study was made on the relatively immediate relaxant effect of cholera toxin (CTX) on the isolated ear artery, thoracic aorta and saphenous vein of the rabbit. Both preparations of CTX, containing sodium azide (NaN3) and azide-free, showed no effect on the non-precontracted artery, but CTX containing NaN3 relaxed the moderately precontracted blood vessels with methoxamine promptly, i.e., with a time course of min order. However, the immediate relaxation produced by CTX containing NaN3 was attributed mainly to NaN3. Azide-free CTX, on the other hand, at 1-10 micrograms/ml gradually produced concentration-dependent relaxation of the precontracted vessels. The relaxant effects of CTX on the vessels were slow and long-lasting, i.e., with a time course of 10 min order. The relaxation induced by CTX was not influenced by the removal of endothelium nor by pretreatment with 10 microM indomethacin, 3 microM atropine or 3 microM propranolol. Activation of protein kinase C by a phorbol ester inhibited the relaxant effect of CTX. These results indicate that CTX relaxes the blood vessels by directly acting on the smooth muscles, without mediation by known endogenous relaxing factor, such as endothelium-derived relaxing factor (EDRF = NO) or prostaglandin I2 (prostacyclin) and by muscarinic receptor or beta-adrenoceptor.