Since clotrimazole, known as an antifungal drug, exerts diverse actions on cellular functions, it is expected that clotrimazole can be used for other purposes. This antifungal drug protects the cells overloaded with Ca(2+) by A23187, a calcium ionophore. Therefore, the agent may prevent the cells from death induced by heavy metals such as CdCl(2), PbCl(2), or HgCl(2) that are respectively proposed to increase intracellular Ca(2+) concentration. To test this possibility, we have examined the effect of clotrimazole on the cells simultaneously treated with CdCl(2), PbCl(2), or HgCl(2) using rat thymocytes and a flow cytometer with fluorescent probes. The simultaneous application of clotrimazole and CdCl(2) significantly decreased cell viability, even though the concentrations of both were ineffective at affecting the viability. The significant decrease in cell viability was not due to the inhibition of Ca(2+)-ATPase and Ca(2+)-dependent K(+) channels that were induced by clotrimazole. The simultaneous application increased the population of cells with phosphatidylserine exposed on membrane surface, indicating the change in asymmetrical distribution of membrane phospholipids. Furthermore, the cytotoxicity induced by the combination of clotrimazole and CdCl(2) under nominally Ca(2+)-free condition was more profound than that under normal Ca(2+) condition. Therefore, the membrane may be a target for the cytotoxic action of clotrimazole and CdCl(2) that were simultaneously applied. It is also the case for PbCl(2), but not the case for HgCl(2). It is concluded that clotrimazole can modulate the cytotoxicity of some heavy metals.