1. The effects of the calmodulin antagonists, calmidazolium and fendiline were investigated on endothelium-dependent hyperpolarization in the canine coronary artery. The membrane potential of vascular smooth muscle cells was measured with the microelectrode technique. 2. Smooth muscle cells of the canine coronary artery had a resting membrane potential of -50 mV. Bradykinin and the Ca(2+)-ionophore, A23187, induced concentration- and endothelium-dependent hyperpolarization. The hyperpolarization induced by a supramaximal concentration of bradykinin (10(-6) M) reached approximately 20 mV. 3. Calmidazolium (10(-5) M) and fendiline (10(-4) M) inhibited hyperpolarization induced by bradykinin and A23187. By contrast, calmidazolium did not affect the hyperpolarization induced by lemakalim, an opener of ATP-sensitive K(+)-channels. 4. These observations suggest that calmodulin is involved in the generation of endothelium-dependent membrane hyperpolarization of vascular smooth muscle.