Protein kinase C (PKC) phosphorylates the catalytic alpha 1 subunit of Na,K-ATPase in purified enzyme preparations and in intact cells. Little is known, however, whether all three known alpha isoforms are substrates for PKC and whether direct phosphorylation is implicated in the modulation of the transport activity of the different Na,K-ATPase isozymes. In this study, we investigated the structural requirements for PKC phosphorylation of alpha 1, alpha 2, and alpha 3 isoforms of different species after expression in Xenopus oocytes. By using a combination of site-directed mutagenesis and computer-assisted protein modeling, we characterized a novel Ser-X-His motif which in concert with more distantly located basic residues acts as an efficient substrate for PKC-mediated phosphorylation in the N-terminus of most Na,K-ATPase alpha 1 isoforms. As indicated by controlled proteolysis, alpha 2 isoforms are also phosphorylated in the N-terminus but to a much lower extent than alpha 1 isoforms containing the Ser-X-His motif. Phosphorylation and phosphoamino acid analysis of fusion proteins containing the wild-type or mutant N-terminus of alpha 2 reveal that Thr-Thr-Ser-X-Asn or Thr-Thr-Ala-X-Asn motifs represent weak targets for PKC phosphorylation. Finally, our data suggest that, with the exception of rat alpha 3, all alpha 3 isoforms from other species are not substrates for PKC. On the basis of the phosphorylation efficiency, we may speculate that only alpha 1 but not alpha 2 or alpha 3 isoforms of Na,K-ATPase are likely candidates for regulatory PKC phosphorylation.