The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca 2 + concentration ([Ca 2 + ] i ), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA-EB fluorescent complex formation was studied in ergosterol-containing Leishmania promastigotes. Intracellular Ca 2 + concentration was measured using quin-2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca 2 + ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca 2 + ] i . On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca 2 + ionophore did not provoke cell penneabilization to EB.