Using conventional and ion selective microelectrodes, the effect of ouabain (10(-4) mol/l) on peritubular cell membrane potential (PDpt), on intracellular pH (pHi) as well as on the intracellular ion activities of Cl- (Cli-), K+ (Ki+), Na+ (Nai+) and Ca2+ (Ca2i+) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PDpt = -57.0 +/- 1.9 mV), the electrochemical potential difference of chloride (apparent mu Cl- = -22 +/- 2 mV) and of potassium (mu K+ = +24 +/- 3 mV) is directed from cell to bath, of H+ (mu H+ = -42 +/- 5 mV), of Na+ (mu Na+ = -102 +/- 4 mV) and of Ca2+ (mu Ca2+ = -148 +/- 6 mV) from bath to cell. Ouabain leads to a gradual decline of PDpt, which is reduced to half (PDpt, 1/2) within 31 +/- 4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent mu Cl+, of mu H+, mu Na+ and mu Ca2+. In contrast, an increase of mu K+ is observed. At PDpt, 1/2 apparent Cl-i increases by 6.2 +/- 1.0 mmol/l, pHi by 0.13 +/- 0.03, Ca2+i by 185 +/- 21 nmol/l, and Nai+ by 34.2 +/- 4.6 mmol/l, whereas Ki+ decreases by 37.7 +/- 2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K+, by an accumulation of Ca2+, Na+ and HCO3- in the cell and by a dissipation of the electrochemical Cl- gradient.