The mechanisms of cellular lead uptake were characterized using a fluorescence method in cells loaded with indo-1. Pb2+ bound to intracellular indo-1 with much higher affinity than Ca2+ and quenched fluorescence at all wavelengths. Pb2+ uptake into pituitary GH3 cells, glial C6 cells, and a subclone of HEK293 cells was assessed by fluorescence quench at a Ca2+-insensitive emission wavelength. Pb2+ uptake was concentration- and time-dependent. Pb2+ uptake in all three cell types occurred at a much faster rate when intracellular Ca2+ stores were depleted by two different methods: addition of drugs that inhibit the endoplasmic reticulum Ca2+ pump (thapsigargin, cyclopiazonic acid, and tert-butylhydroquinone), and prolonged incubation of cells in Ca2+-free media. Application of receptor agonists, which deplete intracellular Ca2+ stores via inositol trisphosphate-sensitive channels, did not activate Pb2+ uptake. Agonists were just as effective as thapsigargin in stimulating uptake of Ca2+ but less so in stimulating uptake of Mn2+. Basal and stimulated Pb2+ uptake were partially reduced by 1 mM extracellular Ca2+ and strongly inhibited by 10 mM Ca2+. Pb2+ entry in GH3 cells was inhibited by two drugs that block capacitative Ca2+ entry, La3+ and SK&F 96365. Depolarization of electrically excitable GH3 cells increased the initial rate of Pb2+ uptake 1.6-fold, whereas thapsigargin increased uptake 12-fold. In conclusion, Pb2+ crosses the plasma membrane of GH3, C6, and HEK293 cells via channels that are activated by profound depletion of intracellular Ca2+ stores.