Peroxynitrite is a nitric-oxide-derived cytotoxic mediator produced in a broad range of inflammatory conditions, ranging from sunburn erythema to contact hypersensitivity. Our previous work has shown that in HaCaT cells the cytotoxic activity of peroxynitrite involves both apoptotic and necrotic routes with poly(ADP-ribose) polymerase activation serving as a mol-ecular switch diverting the default apoptotic pathway toward necrosis. Nonetheless, keratinocytes are regarded as highly resistant toward environmental noxa including oxidative stress. We set out to investigate the possible role of two parameters, intracellular calcium mobilization and high cell density, in protecting HaCaT cells from peroxynitrite/oxidative-stress-induced cytotoxicity. First we characterized the effect of peroxynitrite on the calcium homeostasis of HaCaT cells and demonstrated that both authentic peroxynitrite and the peroxynitrite generating compound 3-morpholino-sydnonimine triggered an elevation in intracellular calcium levels. Moreover, we established that treatment of cells with the cell-permeable calcium chelator BAPTA-AM provided significant cytoprotection against peroxynitrite- and hydrogen-peroxide-induced cytotoxicity. Furthermore, when cells reached confluence they were highly resistant to the toxic effects of peroxynitrite, hydrogen peroxide, and superoxide. The resistance to oxidative stress provided by calcium chelation and high cell density involved inhibiting the activation of both poly(ADP-ribose) polymerase and caspases. Our data may provide an explanation for the resistance to oxidative stress of superficial, highly differentiated keratinocytes and indicate that basal proliferative keratinocytes are sensitive in vivo targets of oxidative stress injury.