Chloro(2,2':6',2"-terpyridine) platinum, a bulky, hydrophilic reagent, inhibited the renal sodium pump with a single exponential time course. K(+) increased the rate constant of the reaction by about twofold; the K(+) concentration dependence was monotonic, with a half-maximal effect observed at 1 mM, consistent with K(+) acting at a transport site. Na(+), Mg(2+), eosin, and vanadate did not significantly alter the rate of reaction. The results of proteolysis and mass spectrometer analysis were consistent with terpyridine platinum labeling of Cys452, Cys456, or Cys457. Because phenylarsine oxide reacts with vicinal cysteines and did not prevent terpyridine platinum modification, terpyridine platinum most likely modifies Cys452. This modification prevents ADP binding; interestingly, the analogous residue in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is on the exterior of the nucleotide-binding pocket. Thus it appears that the terpyridine platinum residue is more accessible in the presence of K(+) than in its absence and that terpyridine platinum modification prevents nucleotide binding.