We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca²⁺ concentration ([Ca²⁺]_i) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca²⁺ mobilization and Na⁺-K⁺-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca²⁺]_i resembled that induced by the Ca²⁺ channel blocker nifedipine and were reversed specifically by the Ca²⁺ channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca²⁺ influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.