Site-directed mutagenesis studies of the structure and function of the Ca2+ binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase are reviewed. The Ca2+ binding properties of six mutants with alterations to amino acid residues with oxygen-containing side chains in the membrane segments M4, M5, M6, and M8 were investigated. The mutations to Glu309 in M4, Glu771 in M5, Asn796, Thr799, and Asp800 in M6 all disrupted Ca2+ occlusion, suggesting that the side chains of these residues donate oxygen ligands to Ca2+ binding at the high-affinity sites and/or are involved in conformational changes that occlude the sites. Alanine substitution of Glu908 in transmembrane segment M8 did not prevent Ca2+ occlusion, thereby excluding this residue from playing a central role in Ca2+ coordination. Titrations of Ca2+ activation of phosphorylation from ATP and of inhibition by Ca2+ of phosphorylation from Pi allowed us to assign Ca2+ liganding residues separately to the two high-affinity Ca2+ sites. Hence, residues Glu771 and Thr799 are associated with the site binding the first calcium ion in the sequential mechanism ("site 1"), whereas Glu309 and Asn796 are associated with the site binding the second calcium ion ("site 2"), and Asp800 donates Ca2+ ligands to both sites. On this basis we discuss two possible structural models for the Ca2+ sites.