Our purpose was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X₇ and P2Y₄ purinoceptors, this nucleotide can kill retinal microvascular cells. Because P2X₇ receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for the lethality of ATP. To test this hypothesis, we isolated pericyte-containing microvessels from rat retinas and assessed cell viability by trypan blue dye exclusion, detected pores by the uptake of the fluorescent dye YO-PRO-1, measured intracellular calcium with the use of fura-2 and monitored ionic currents via perforated-patch pipettes. As predicted, ATP-induced cell death required P2X₇ receptor activation. However, we found that pore formation was minimal because the activation of P2Y₄ receptors by ATP prevented P2X₇ pores from forming. Rather than opening lethal pores, ATP kills by a mechanism involving voltage-dependent calcium channels (VDCCs). Our experiments suggest that when high concentrations of ATP cause nearly all microvascular P2X₇ receptor/channels to open, the resulting profound depolarization opens VDCCs. Consistent with VDCCs accounting for a lethal calcium influx, ATP-induced cell death was markedly diminished by the VDCC blocker nifedipine or a nitric oxide (NO) donor that inhibits microvascular VDCCs. We propose that purinergic vasotoxicity is normally prevented in the retina by an NO-mediated inhibition of VDCCs and a P2Y₄-mediated inhibition of P2X₇ pore formation. Conversely, dysfunction of these protective mechanisms may be a previously unrecognized cause of cell death within the retinal microvasculature.