Two recent studies have demonstrated that clotrimazole, a well-known potential antifungal agent, inhibits the in vitro growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum. In a previous study, we suggested that clotrimazole acts as an anti-malarial agent by inhibiting heme catabolism in the malaria parasite and by enhancing heme-induced membrane damage. In this paper, we examined the mechanism of action by measuring hemolysis as an indicator of membrane damage. Our results showed that clotrimazole does not promote the binding of heme to membranes, and that the enhancement of heme-induced hemolysis by clotrimazole is not caused by lipid peroxidation or by oxidation of thiol groups in membrane proteins. Instead, clotrimazole inhibits glutathione-dependent heme degradation, resulting in an enhancement of heme-induced hemolysis. We also found that clotrimazole increases the susceptibility of erythrocytes to hypotonic lysis in the presence of heme and that sucrose could inhibit hemolysis induced by heme-clotrimazole complexes. Thus, it appears that the enhancement of heme-induced hemolysis by clotrimazole in our experiments is due to a colloid osmotic hemolysis mechanism. The hydrophobicity and the large molecular size of the heme-clotrimazole complex might be key factors for induction of hemolysis.