Changes in intracellular Ca2+ concentration ([Ca2+]i) are believed to have a crucial role in triggering apoptosis, the process whereby cells are eliminated during tissue turnover, hormone-mediated tissue reabsorption, clonal selection in the thymus, T cell-mediated cytotoxic reactions, and many other physiological and pathological processes. In the present report we have explored the role of intracellular Ca2+ and Zn2+ in the regulation of apoptosis in peripheral blood lymphocytes (PBL). Unless they were stimulated by a mitogen, e.g., phytohemagglutinin (PHA), PBL showed typical apoptotic changes (nuclear condensation and DNA fragmentation) after a 48- to 72-h culture. Neither a sustained nor a transient increase in [Ca2+]i induced by the Ca2+ ionophore ionomycin in the presence or absence of extracellular Ca2+, respectively, was able to trigger DNA fragmentation. On the contrary, [Ca2+]i chelation to a level 10- to 20-fold lower than physiological resting levels induced DNA cleavage into nucleosome-size DNA fragments. DNA cleavage was also induced by incubation in the presence of the intracellular, membrane-permeant Zn2+ chelator N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine. Total intracellular Zn2+ content was increased by mitogenic stimulation with PHA and decreased by treatment of PBL cultures with dexamethasone or extracellular ATP, stimuli known to trigger apoptosis in lymphocytes. Our results suggest that Zn2+ rather than Ca2+ could be a key intracellular regulator of apoptosis.