We describe the results of a study designed to identify cDNAs encoding Ca2+-transporting ATPases and other cation-transporting ATPases of the aspartylphosphate class. Rat brain, kidney, and stomach cDNA libraries were screened with an oligonucleotide hybridization probe corresponding to a 23-amino acid sequence from part of the ATP-binding site of the sarcoplasmic reticulum Ca-ATPase. This procedure resulted in the isolation of cDNAs encoding (i) the plasma membrane Ca-ATPase, (ii) an apparent Ca-ATPase that exhibits high amino acid similarity to the sarcoplasmic reticulum Ca2+ pumps, (iii) a transport ATPase of unknown ion specificity and (iv) two Ca-ATPase isoforms encoded by the gene for the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase. Several isoforms of the Na,K-ATPase and gastric H,K-ATPase that had been characterized previously were also identified. The complete nucleotide sequences have been determined for the two classes of cDNA derived from alternatively spliced transcripts of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene. One of these cDNAs, isolated from the stomach library, encodes a Ca-ATPase that is identical to the skeletal muscle enzyme. The second class of cDNA, found in brain, kidney, and stomach libraries, is identical to that of the slow-twitch isoform throughout much of its length but encodes an alternative C terminus and has a different 3'-untranslated sequence. Whereas the muscle isoform consists of 997 amino acids and terminates with the sequence Ala-Ile-Leu-Glu, the second isoform is 1043 amino acids in length due to the replacement of these last 4 amino acids with a 50-amino acid sequence that contains a potential transmembrane domain followed by a consensus sequence for an N-linked glycosylation site.