To explore the function of protein kinase C in smooth muscle, the effects of phorbol esters, potent activators of protein kinase C, were examined in guinea-pig tracheal rings and ileal strips. In tracheal rings, phorbol-12,13-diacetate (PDA) and 12-deoxyphorbol-13-isobutyrate (DPB), both potent stimulants of protein kinase C, produce a concentration dependent, reversible relaxation of resting tracheal tension, whereas phorbol, an inactive analogue is ineffective. PDA also reverses contractions produced by carbachol, serotonin, prostaglandin F2 alpha and prostaglandin D2. In contrast to their ability to inhibit agonist induced contractions, PDA and DPB greatly amplify the constriction produced by a depolarizing concentration of KCl (59 mM). The calcium channel blockers verapamil, nifedipine and diltiazem block the constriction produced by KCl and PDA suggesting that under depolarizing conditions, PDA synergizes with increased intracellular calcium to potentiate muscle contraction. Similar biphasic responses to phorbol esters are elicited in strips of guinea-pig ileum. These results indicate that in addition to enhancing the actions of intracellular calcium in producing contraction, protein kinase C can also activate feedback mechanisms which limit cellular responses.