Zinc ions are essential, but at elevated concentrations, they also have toxic effects on mammalian cells. Zinc plays a crucial role in cell proliferation and differentiation and it even protects cells against apoptosis caused by various reagents. On the other hand, zinc at high concentrations causes cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, and breakdown of the mitochondrial membrane potential. In the present work, a clone of rat C6 glioma cells that was resistant to toxic effects of ZnCl2 up to 250 microM was employed to study the effect of the ionophore A23187 on zinc-induced apoptosis. Neither 150 microM Zn2+ nor 100 nM A23187 alone caused apoptosis as measured by internucleosomal DNA fragmentation. However, combined exposure of C6 cells to 100 nM A23187 and 150 microM Zn2+ for 48 h was effective in inducing apoptosis. Because the so-called calcium ionophore A23187 is not specific for Ca2+ ions but also transports Zn2+ with high selectivity over Ca2+, we investigated whether this substance promoted the uptake of Zn2+ ions into C6 cells. Employing the zinc-specific fluorescence probe Zinquin, we observed that the very low concentration of 1.9 nM A23187 significantly and rapidly raised the intracellular mobile Zn2+ content. Analysis by atomic absorption spectroscopy revealed that incubation with 1.9 nM A23187 caused a doubling of the total intracellular zinc level within 60 min. We conclude that the apoptosis evoked by the combined action of Zn2+ and A23187 was the result of enhanced Zn2+ influx evoked by the ionophore, resulting in higher intracellular zinc levels.