Apoptosis is a pathway of cell death characterized by internucleosomal digestion of genomic DNA. Such DNA digestion can be induced by both physiological stimuli and cytotoxic treatment with many anticancer agents. This digestion has generally been considered to be mediated by a Ca2+/Mg(2+)-dependent endonuclease that is activated by increases in intracellular Ca2+. However, we suggest that an alternate endonuclease, DNase II, may be a more likely candidate. In these studies, apoptosis was induced in human HL-60 cells by a 30-min incubation with the topoisomerase II inhibitor etoposide. DNA digestion characteristic of apoptosis began within 3 h of removal of etoposide. Morphological indication of apoptosis was observed concurrently. Only about 20% of the cells underwent apoptosis at this time; these appeared to be cells in S phase at the time of etoposide treatment. The remainder of the cells progressed to the G2 phase and arrested there for at least 48 h. Intracellular Ca2+ and pH were measured in individual cells by flow cytometry. No changes in intracellular Ca2+ were observed, but an acidification of up to 1 pH unit occurred in about 15% of the cells and correlated with the time course of appearance of DNA digestion. Cells were sorted on the basis of intracellular pH and only the acidic cells showed the morphology and DNA digestion characteristic of apoptosis. These results demonstrate the involvement of DNase II in apoptotic DNA digestion and suggest mechanisms of pH homeostasis as regulators of apoptosis.