A new P-type ATPase gene, cta3, has been identified in Schizosaccharomyces pombe. The deduced amino acid sequence presents a 45% identity with the Saccharomyces cerevisiae putative Ca2(+)-ATPase encoded by the PMR2 gene. The cta3 protein contains 7 out of the 8 amino acid residues involved in high affinity Ca2+ binding in the sarcoplasmic reticulum Ca2(+)-ATPase from muscles. It also contains a region similar to the phospholamban-binding domain that characterizes this Ca2+ pump. A null mutation of cta3 leads to higher levels of cytosolic free Ca2+ and to lower amounts of sequestered and bound Ca2+. Cellular Ca2+ efflux and rates of uptake into intracellular compartments are reduced by the loss of cta3 function. The sequence analysis and the physiological results strongly support the conclusion that the cta3 gene encodes a Ca2(+)-ATPase, probably located in intracellular membranes.