A novel pyridine derivative, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidonopyridine hydrochloride, AU-1421, was found to produce reversible inhibition of the dog kidney sodium and potassium ion-dependent adenosine triphosphatase [(Na,K)-ATPase] with I50 values of about 50 microM. The reversible inhibition was observed when the enzyme was added directly to the enzyme assay media in the presence of saturating concentrations of the enzyme ligands, Na+, K+, Mg2+ and ATP ("turnover conditions"). In the present study, we focused on the reversible inhibition without preincubation of the enzyme with AU-1421. This inhibition was competitive with respect to K+. The K(+)-pNPPase activity of the same preparation was also inhibited by AU-1421 with I50 values of about 90 microM, and this manner was also competitive with respect to K+. ATP enhanced the AU-1421 inhibition of (Na,K)-ATPase, suggesting that AU-1421 also bound to the enzyme-substrate complex. AU-1421 inhibition of (Na,K)-ATPase was not antagonized by ouabain, suggesting the difference of the binding sites between AU-1421 and ouabain. It is therefore proposed that AU-1421 reversibly interacts at or near the K+ site during turnover conditions.