Experiments were undertaken to investigate the role of intracellular Ca'+ stores in the regulation of Ca2+ uptake in the cultured B-lymphocytic cell line CH12.LX.C4.5F5. Release of intracellular C a l f stores by addition of thapsigargin was accompanied by a biphasic increase in intracellular calcium concentration [Ca"],). The initial rise in [Ca2 'Ii was due to release of CaLt from intracellular stores as determined by its maintenance in the absence of extracellular Ca2+. The secondary phase was 1) dependent on the presence of extracellular Ca2+, 2) inhibited by 5 m M extracellular Ni2+, and 3) inhibited by high K' , consistent with electrogenic Ca2+ uptake from the extracellular medium. In order to more accurately investigate the electrogenic nature of this pathway we measured the membrane potential changes accompanying Ca2+ influx stimulated by release of Ca2+ from intracellular stores using bis(l,3-diethylthiobarbituric acid trimethine) oxonol in Bapta-loaded cells. Addition of 5 m M Ca2+ to cells pretreated with doses of thapsigargin or ionomycin shown to release intracellular CaL+ stores induced a depolarization which was 1 ) dependent upon extracellular Ca2+, 2) abolished by 5 m M Nil+, 3) independent of extracellular Na+, and 4) dependent upon Bapta loading. This depolarization was followed by a charybdotoxin-sensitive repolarization consistent with secondary activation of K+ channels. Changes in [Ca2+Ii monitored under identical conditions were monitored fluorimetrically using indo-1 and were found to correlate with the changes in Em. On the basis of these data we conclude that an electrogenic Ca2+-permeable pathway exists in this B-lymphocytic cell line which is regulated by the degree of filling of an internal CaL+-StOre.