1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]-nimodipine binding. Trifluoperazine and W-7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation. 4. Ba2+ current flowing through voltage-gated Ca2+ channels was measured under whole-cell voltage-clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration-range where inhibition of [3H]-nimodipine binding was observed. Neither voltage-dependence nor the inactivation time course of Ba2+ current were affected by these compounds. 5. The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W-7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds.