Friend murine erythroleukemia cells (MEL cells) contain a cAMP-independent protein kinase which phosphorylates the 100,000-Da catalytic subunit of the (Na,K)-ATPase both in living cells and in the purified plasma membrane (Yeh, L.-A., Ling, L., English, L., and Cantley, L. (1983) J. Biol. Chem. 258, 6567-6574). We have taken advantage of the selective phosphorylation of the 100,000-Da subunit in purified plasma membranes and the similarity between the proteolysis patterns of the MEL cell and dog kidney (Na,K)-ATPase to map the site of kinase phosphorylation on the MEL cell enzyme. The chymotryptic and tryptic cleavage sites of the dog kidney (Na,K)-ATPase have previously been located (Castro, J., and Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228). The 100,000-Da catalytic subunits of the dog kidney and MEL cell enzymes were specifically labeled at the active site aspartate residue by incubation with (32P)orthophosphate in the presence of Mg2+ and ouabain. Digestion of these two enzymes with chymotrypsin or trypsin revealed similar active site aspartate containing proteolytic fragments indicating a similar structure for the two enzymes. Chymotryptic digestions of MEL cell (Na,K)-ATPase labeled in vitro with [gamma-32P]ATP localize the region of kinase phosphorylation to within a 35,000-Da peptide derived from the middle of the 100,000-Da subunit. Tryptic digestion of the MEL cell plasma membranes degraded the 100,000-Da subunit to an NH2-terminal 43,000-Da peptide which contained the active site aspartate but which did not contain the kinase-labeled region. These results further locate the region of kinase phosphorylation to the COOH-terminal half of the 35,000-Da chymotryptic peptide. This location places the site of phosphorylation between the active site aspartate residue which accepts the phosphate of ATP during turnover and an ATP-binding site which has previously been located by labeling with fluorescein 5'-isothiocyanate (Carilli, C. T., Farley, R. A., Perlman, D. M., and Cantley, L. C. (1982) J. Biol. Chem. 257, 5601-5606). Phosphorylation of the (Na,K)-ATPase in this region may serve to regulate the activity of this enzyme.