The relative ability of acetylcholine and 5-hydroxytryptamine (5-HT) to contract the vascular smooth muscle of bovine ventricular coronary arteries by mobilizing extracellular calcium was investigated. Methysergide and atropine specifically inhibited contractions to 5-HT and acetylcholine, respectively. Acetylcholine produced a sustained increase in calcium influx and a relatively sustained contraction. Contractions produced by 5-HT have previously been shown to be more transient than those by acetylcholine, and 5-HT increases calcium influx only transiently. The contraction produced by acetylcholine, but not that produced by 5-HT, was inhibited by 1 microM diltiazem to a level not different from that produced in Ca-free physiological saline solution. Verapamil at 0.1 microM did not inhibit an acetylcholine contraction. Steady-state tension produced by KCl was greatly inhibited by 1 microM diltiazem and 0.1 microM verapamil. Force produced in a calcium-free medium by acetylcholine and 5-HT was not additive. After depletion of the agonist-releasable intracellular calcium pool, however, force produced by acetylcholine plus 5-HT in the presence of extracellular calcium was additive. Likewise, calcium influx produced by both agents together was significantly greater than that produced by either agent alone. These results suggest that, in the smooth muscle of bovine ventricular coronary arteries, 5-HT and acetylcholine do not operate the same calcium channels.