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Nineteen mutants that lost the ability to grow in 100 mM Ca2+ (but remained insensitive to 50 mM Sr2+) were identified in a screen of approximately 60,000 mutagenized yeast colonies. Cells carrying mutations in the CSG2 gene grow normally in low Ca2+ medium but have decreased growth rates when the Ca2+ concentration is above 10 mM. The csg2 mutant cells accumulate much higher levels of Ca2+ in a compartment that is exchangeable with extracellular Ca2+ but the nonexchangeable Ca2+ pool which predominates in wild-type cells is not influenced. Sr2+ influx is not increased in the csg2 mutant cells. Mg2+ decreases the amount of Ca2+ in the nonexchangeable pool without influencing the csg2-induced exchangeable Ca2+ pool. The data indicate that the csg2 mutation causes a selective increase in Ca2+ accumulation into a pool which is distinct from the vacuolar pool. The CSG2 protein consists of 410 amino acids, contains nine putative transmembrane segments, four potential sites for N-linked glycosylation, and a sequence with homology to the EF-hand Ca2+-binding site.