The possibility has been examined that Na+-dependent K+-conductive pathways, known to exist in certain excitable cell membranes and inhibitable by the drug R56865, may also be present in cells of rat renal inner medulla, and that their activation may explain aspects of volume regulation by these cells in tissue slices exposed to strongly hyperosmotic media, as reflected by the rate of efflux of preloaded 86Rb+ (a marker for K+) and steady-state cell volumes and K+ contents. Cells incubated in media of 2000 mosmol/kg (400mM Na+, 1172mM urea) shrink and lose K+ by comparison with those in 720 mosmol/kg (203mM Na+, 266mM urea). If 2-aminisobutyric acid (10mM) is added there is partial restoration of cell volume due to inwardly directed Na+-amino acid cotransport, but 86Rb+ efflux is accelerated and cells fail to regain net K+. R56865 (5μM) completely blocks the increase in efflux and causes marked increases in cell volume and K+ contents, but only in strongly hyperosmotic media and in the presence of both Na+ and amino acid. In mildly hyperosmotic media, or media of 2000 mosmol/kg from which Na+ or amino acid is omitted, R56865 is without effect on these variables.