The present study investigates the requirement for cellular ATP in the increase in plasma membrane Ca2+ permeability activated by the release of Ca2+ from intracellular stores in rat thymic lymphocytes (capacitative Ca2+ entry). The permeability state of this pathway following activation with thapsigargin was probed in control and ATP-depleted cells using fluorometric measurements of intracellular Ca2+, Mn2+ entry, and membrane potential, and unidirectional measurements of Ca2+ uptake using 45Ca2+. The capacitative Ca(2+)-entry pathway was markedly inhibited in cells depleted of ATP by incubation in glucose-free solution containing oligomycin, antimycin A, and 2-deoxy-D-glucose. These data cannot be explained on the basis of a loss of the transmembrane electrochemical gradient for Ca2+, alterations in intracellular pH or cellular Na+ content, a direct effect of the inhibitors of ATP production on the capacitative Ca(2+)-entry pathway, or the ability of thapsigargin to release Ca2+ from intracellular stores. Rather, these data are consistent with a requirement for ATP or a high-energy phosphate donor in the activation and/or maintained activation of capacitative Ca2+ entry.