[alpha-32P]-8-N3-ATP, [2-3H]-8-N3-ATP, and non-radioactive 8-N3-ATP have been used as photoaffinity probes of the ATP binding site of dog kidney Na+/K(+)-ATPase. 8-N3-ATP has previously been shown to bind to Na+/K(+)-ATPase with high affinity, to be a substrate for Na+/K(+)-ATPase, and to inactivate the enzyme upon ultraviolet irradiation [Scheiner-Bobis, G., & Schoner, W. (1985) Eur. J. Biochem. 152, 739-746]. 8-N3-ATP competitively inhibits the high-affinity binding of [2,8-3H]-ATP to Na+/K(+)-ATPase with a Ki of 3.4 microM, which is comparable to the reported KD of 3.1 microM for the binding of 8-N3-ATP to the enzyme. The extent of inhibition of ATP hydrolysis by 8-N3-ATP was linearly correlated with the stoichiometry of covalent incorporation of 8-N3-ATP into Na+/K(+)-ATPase up to about 50% inhibition of activity; however, the linkage between the protein and 8-N3-ATP was unstable, and the maximum incorporation of 8-N3-ATP was less than the nucleotide binding capacity of the protein. After photolysis with ultraviolet light, 8-N3-ATP was specifically incorporated into the carboxy-terminal 58-kDa fragment of the alpha-subunit of Na+/K(+)-ATPase generated by limited trypsin digestion in the presence of KCl, and the beta-subunit was not labeled. 8-N3-ATP-labeled Na+/K(+)-ATPase was digested with trypsin, and a single peak containing the nucleotide was identified after HPLC fractionation of the digest.(ABSTRACT TRUNCATED AT 250 WORDS)