To estimate the early effect of aldosterone on the number of active Na(+)-K+ pumps at the basolateral membrane of amphibian tight epithelia, we have measured the initial rate (at 4 min) of [3H]ouabain binding to the basolateral membrane of intact monolayers of A6 cells grown on permeable supports. Within 3 h, aldosterone induced a threefold increase of the Na transport and, simultaneously, a twofold increase of the binding rate of ouabain. Because the affinity of ouabain, estimated either by equilibrium binding studies or inhibition kinetics, was not modified by aldosterone, the effect on the initial rate of ouabain binding was due to an increase in the number of binding sites. This effect on ouabain binding was not prevented by 10 microM amiloride, which reduced the transepithelial sodium transport below control level. By contrast, the effect of aldosterone on ouabain binding was abolished by cycloheximide (5 micrograms/ml) or actinomycin D (2 micrograms/ml), doses which inhibited the aldosterone-dependent sodium transport response. These data suggest that aldosterone elicits an early, sodium-independent, protein synthesis-dependent increase in the expression of active Na(+)-K(+)-ATPase molecules.