Na(+)/K(+)-ATPase plays a key role in the transport of Na(+) throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na(+) and K(+) ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na(+)/K(+)-ATPase activity and K(+) conductance were evaluated using electrophysiological methods. Na(+)K(+)-ATPase alpha(1)- and beta(1)-subunit expression was quantified by Western blot techniques. Na(+)/H(+) exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na(+) transport, which results from an increase in the number of Na(+)/K(+)-ATPase alpha(1)- and beta(1)-subunits, in the membrane. Increases in Na(+)/K(+)-ATPase activity were accompanied by increases in K(+) conductance as a result of functional coupling between Na(+)/K(+)-ATPase and basolateral K(+) channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na(+)/H(+) exchanger activity were observed. H(2)O(2) production was increased in aged cells, but exposure for 5 days to 1 and 10 microM: of H(2)O(2) had no effect on Na(+)/K(+)-ATPase expression. Ouabain (100 nM: ) increased alpha(1)-subunit, but not beta(1)-subunit, Na(+)/K(+)-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.