To determine whether multiple forms of the Na+,K+-ATPase beta subunit exist in rat kidney and brain, we constructed cDNA libraries from both tissues and isolated clones carrying inserts coding for this subunit. Characterization of these cDNAs by restriction endonuclease mapping and DNA sequencing suggests that a single form of the beta subunit protein is present in these tissues. We did, however, find that the beta subunit was encoded by multiple mRNAs which can be grouped into classes based on the lengths of their 5' and 3' untranslated regions. Size variation at the 3' end is due to the use of at least two of the five potential polyadenylation sites, whereas variation at the 5' end appears to be due to the use of different initiation sites. Northern blot analysis demonstrates the presence of multiple mRNA species which correlate with the sizes observed for the different cDNA classes and also demonstrates that the larger mRNA species are present at different levels in kidney and brain. S1 and primer extension analyses suggest that the observed tissue differences in the expression of these mRNAs are due to transcription from multiple initiation sites, whose strength is tissue-dependent. In the 3' untranslated region, comparison of the nucleotide sequence of the rat beta subunit cDNAs to those from other species reveals regions of high sequence similarity.