Extracellular ATP (ATPo) caused a concentration-dependent lysis of mouse thymocytes. Lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase, was preceded by depolarization of the plasma membrane and by Ca2+ influx. Both Na+ uptake (which sustained plasma membrane depolarization) and Ca2+ influx showed (1) the same dependence on the ATPo concentration; (2) the same nucleotide specificity; and (3) the same Hill coefficient. However, whereas the rise in the cytosolic free Ca2+ concentration ([Ca2+]i) was fully inhibited by the known Ca2+ blocker verapamil, plasma membrane depolarization was enhanced under these conditions. Plasma membrane depolarization was greater and was shifted to lower ATPo concentrations in the absence of extracellular Ca2+ (Ca2+o), whereas the rise in [Ca2+]i was greater in Na(+)-free media. Plasma membrane depolarization also occurred in Na(+)-free choline- or methylglucamine-containing media, and was potentiated by chelation of free divalent ions with EDTA, supporting previous reports pointing to ATP4-as the active species. Among a number of purine and pyrimidine nucleotides, only adenosine 5'-[gamma-thio]triphosphate and ADP were partially effective. Furthermore, ethidium bromide (Mr 380), Lucifer Yellow (Mr 463) and Eosin Yellowish (Mr 692) did not permeate through the ATPo-activated channel. These findings suggest that lytic effects of ATPo in mouse thymocytes depend on the activation of a membrane channel with low selectivity for cations and an Mr cut-off of 200.