The positions, with respect to the plasma membrane, of lysine 905, contained in the peptide QRKIVE, and of lysine 1012, contained in the carboxy-terminal peptide, RPGGWVEKETYY, of ovine Na+/K(+)-transporting ATPase have been reported to be cytoplasmic and extracytoplasmic, respectively [Bayer, R. (1990) Biochemistry 29, 2551-2256]. These results from our laboratory have been reexamined using an extension of the same procedure. Sealed right-side-out vesicles were modified with pyridoxal phosphate and sodium [3H]borohydride in the presence and absence of saponin or cholate. The modified alpha polypeptide was isolated and digested with the proteinase from Staphylococcus aureus strain V8 or trypsin to produce one or the other of these two peptides. These digests were passed over immunoadsorbents, identical to those used by Bayer, directed against pyroglutamylRXIVE or -ETYY. Unlike in the earlier studies, however, in the present studies the modified, radioactive peptides bound and eluted from the immunoadsorbents were submitted to HPLC, and their respective mobilities were compared to those of the synthetic peptides that had also been modified with pyridoxal phosphate. In this manner, the correct, modified peptide could be positively identified, and its specific radioactivity could be estimated. When cholate was added to sealed vesicles, prior to modification, there was at least a 3-fold increase in the incorporation of radioactivity into lysine 1012, consistent with a cytoplasmic location for this residue.(ABSTRACT TRUNCATED AT 250 WORDS)