The regulation of cellular cytotoxicity induced by hydrogen peroxide (H2O2) over a wide concentration range was assessed. Three distinct patterns were detected: the highest concentrations (> 10 mM) rapidly induced a necrotic form of death characterized by smeared patterns of DNA digestion and morphological evidence of primary cytoplasm and plasma membrane damage; In contrast, 10 and 5 mM H2O2 induced endonucleosomal DNA digestion concurrently with cytotoxicity and target cell death was associated with morphologic evidence of apoptosis. Apoptosis was inhibited by cycloheximide, emetine, aminobenzamide (ABA), aurintricarboxylic acid, and calcium depletion. The lowest concentrations of H2O2 (0.5 and 0.1 mM)-induced delayed cytotoxicity (at 24 or 48 hr), which was not associated with DNA ladder formation or morphologic evidence of apoptosis, but was inhibited by ABA. Enforced expression of BCL-2 induced resistance to 0.5 and 0.1 mM H2O2 but had no effect on cytotoxicity induced by 5 and 10 mM. Exposure of isolated nuclei to H2O2 in the absence of calcium or magnesium failed to induce endonucleosomal fragmentation. These data indicate that distinct pathways of H2O2-induced cytotoxicity can be distinguished by their different concentration dependences, and that BCL-2 can protect against some forms of H2O2-induced cytotoxicity.