Polyclonal antibodies raised against the alpha- and beta-subunits of amphibian kidney Na+-K+-ATPase were used to screen an expression library from Xenopus laevis kidney epithelial cells (A6 cell line). cDNAs coding for each Na+-K+-ATPase subunit were identified and used to isolate near full-length cDNAs. The complete nucleotide sequence and the deduced amino acid sequence were determined. The alpha-subunit is an alpha (alpha I)-isoform. The alpha- and beta-subunits are more closely related to the mammalian and avian than the fish sequences. Antibodies raised against the fusion proteins produced by the two clones served to immunoprecipitate proteins from biosynthetically labeled or selectively surface-radioiodinated A6 cells grown on a porous substrate. The alpha- and the beta-subunits of Na+-K+-ATPase were found associated early in the course of biosynthesis and were restricted to the basolateral plasma membrane.