The effects of the Staphylococcus aureus leukocidin (PVL), a two-component non-hemolytic toxin, were investigated on the membrane permeability of human polymorphonuclear leukocytes (PMNs). In the absence of extracellular Ca²⁺, the fluorescence of ethidium bromide added to the extracellular medium increased after PVL application in a concentration-dependent manner and no variations in the free intracellular [Ca²⁺] of Fura2-loaded PMNs were detected. In the presence of extracellular Ca²⁺, the fluorescence of ethidium was not modified but the free intracellular [Ca²⁺] of PMNs increased after application of PVL in a concentration-dependent manner. The time lag observed an increase in the ethidium fluorescence was longer than the time lag observed a Fura2 fluorescence increase. Simultaneous recordings of the two probes fluorescence variations have shown the protective effect of Ca²⁺ and Zn²⁺ and the closing of the pore by 50 mM Ca²⁺ or 2 mM Zn²⁺. Moreover, the effect of Ca²⁺ could be reversed by the addition of EGTA. In the presence of 1 mM extracellular Ca²⁺ or 0.8 mM extracellular Zn²⁺, the pore induced by PVL had an ionic size allowing Ca²⁺, Mn²⁺ and Mg²⁺ fluxes. The addition of antibodies against either compound of PVL inhibits the permeabilization provoked by the toxin even after it was initiated. It is concluded that leukocidin from S. auerus is a pore-forming toxin which, under physiological conditions ([Ca²⁺] = 1 to 1.5 mM), provokes the formation of an ion-sized pore including an increase in the free intracellular Ca²⁺ which may activate PMN functions.