Na,K-ATPase in the retinal pigment epithelium (RPE) is apically localized, whereas in most other tissues this pump is found predominantly in the basolateral membrane domain. As part of our investigations into the molecular aspects of this pump in the RPE, we have cloned the cDNA and characterized the expression of the gene encoding the beta 2 subunit isoform of Na,K-ATPase in human, rat and bovine RPE and in the bovine choroid plexus. We have also detected the beta 2 isoform polypeptide in the human RPE (hRPE). Comparison of complete coding sequences derived from cloned cDNAs revealed that all beta 2 sequences from RPE, and the choroid plexus, differed uniformly at positions: P51/L, M121/I, and L148/R from the published sequences for human retina and liver. However, analysis of 10 RT-PCR clones derived from 5 fetal and 2 adult human retinas sequenced in our laboratory, revealed that only the P51/L residue was different with the hRPE beta 2 subunit sequence. Northern blot analysis indicated a 3.4-kb RNA transcript for the beta 2 subunit, a 4.5-kb RNA for the alpha 1 subunit and a doublet of 2.3 and 2.6 kb for the beta 1 subunit, respectively. alpha 1 (100 kDa), beta 1 (45 kDa) and beta 2 (65 kDa) isoforms were detected in hRPE extracts by immunoblotting. No alpha 2 and alpha 3 RNA transcripts were found in the hRPE. Quantification of beta 2 mRNA by RT-PCR revealed 2.7 x 10(5) molecules per ng of poly A+ RNA. This is similar to the beta 1 isoform levels reported previously from our laboratory. These data demonstrate the coexistence of significant amounts of alpha 1, beta 1 and beta 2 Na,K-ATPase subunits in the RPE. It is therefore reasonable to suggest that both alpha 1 beta 1 and alpha 1 beta 2 heterodimers are present in these cells.