Lithium is the primary treatment for mania, and it has been employed as a therapeutic agent in the treatment of a number of other conditions. In spite of its widespread clinical use, the specific mechanism by which lithium acts is still not known. Because lithium bears some chemical resemblance to Ca2+ and because it has been found to interfere with many Ca2+-dependent processes, we investigated the possibility that lithium can alter intracellular Ca2+ homeostasis by an effect on Ca2+ fluxes. When added in vitro to synaptosomes prepared from rat forebrains, lithium had no effect on 45Ca2+ influx mediated by fast or slow phase depolarization-dependent Ca2+ channels or by Na+/Ca2+ exchange. In vitro treatment with lithium also had no effect on ATP-dependent Ca2+ sequestration by mitochondria or synaptosomal endoplasmic reticulum. In contrast, 45Ca2+ influx in synaptosomes prepared from rats treated chronically with lithium was increased significantly relative to controls. The fact that chronic in vivo treatment with lithium affected synaptosomal Ca2+ flux whereas in vitro treatment did not is consistent with the time course of therapeutic effectiveness in man. The results suggest that lithium does not directly interact with synaptosomal Ca2+ flux, but rather may influence Ca2+ flux through an indirect mechanism following chronic treatment.