Complementary DNAs for two isoforms of the plasma membrane Ca2+-ATPase from rat brain have been isolated. The cDNAs were identified using an oligonucleotide probe derived from a conserved amino acid sequence of the ATP binding site of the aspartylphosphate family of transport ATPases. The complete nucleotide sequences have been determined, and the primary structures for both isoforms have been deduced. The Mr of isoform 1, which has 1,176 amino acids, is 129,500 and that of isoform 2, which has 1,198 amino acids, is 132,605. A region of isoform 1 exhibits perfect amino acid identity with a fragment consisting of 17 amino acids from the phosphorylation domain of the human erythrocyte calmodulin-sensitive Ca2+-ATPase (James, P., Zvaritch, E.I., Shakhparonov, M.I., Penniston, J.T., and Carafoli, E. (1987) Biochem. Biophys. Res. Commun. 149, 7-12). A comparison of transport ATPases from diverse species has allowed the identification of a sequence that may form part of a second ATP binding site. Amino acid similarity and hydropathy profile comparisons suggest that the transmembrane organization of the plasma membrane Ca2+-ATPase is the same as that of the Na+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase; the data indicate that each of these enzymes has an even number of transmembrane domains and that their C termini are located on the cytoplasmic side of the membrane. An arginine-rich sequence that is highly characteristic of the "A" domain of a calmodulin binding site is located near the C termini of both isoforms. This putative A domain is identical in isoforms 1 and 2 but their "B" domains, and the remaining C-terminal sequences, are very different. However, 3'-untranslated sequences of the isoform 1 transcript have the potential to encode a calmodulin binding B domain and a C terminus that is very similar to that of isoform 2. This suggests the possibility that additional diversity may occur via alternative processing of the primary transcript.