Extracellular ATP (ATP(o)) 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 ATP(o) 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 ATP(o) 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 (M(r) 380), Lucifer Yellow (M(r) 463) and Eosin Yellowish (M(r) 692) did not permeate through the ATP(o)-activated channel. These findings suggest that lytic effects of ATP(o) in mouse thymocytes depend on the activation of a membrane channel with low selectivity for cations and an M(r) cut-off of 200.