To investigate the mechanism by which ouabain causes acute increase in peripheral resistance, the effect of ouabain on vessel tone, 22Na efflux and membrane potential has been compared with the binding of [3H]ouabain in rat isolated mesenteric resistance vessels (lumen diameter approximately 200 microns). Ouabain at concentrations between 1 X 10(-8) and 1 X 10(-3) M had no effect on the tone of resting vessels but caused potentiation of the tone of vessels which were submaximally contracted with noradrenaline. In the entire concentration range, these acute potentiating effects of ouabain on vessel tone correlated well with an acute inhibitory effect on 22Na efflux and with acute [3H]ouabain binding, consistent with the vasoconstrictor effect of ouabain being associated with inhibition of the Na-K pump. At all concentrations, in both resting and activated vessels, ouabain exposure caused an acute concentration-dependent depolarization of the membrane. At low ouabain concentrations (1 X 10(-6) and 1 X 10(-5) M) the potentiating effect on vessel tone and the inhibitory effect on 22Na efflux were transient, so that after 10 min no effect of ouabain was seen. This was consistent with these ouabain concentrations failing to cause a measurable increase in the intracellular Na concentration ([Na]i) after longer term (up to 2 h) exposure. The recovery of 22Na efflux was due to a recovery of the ouabain-sensitive part of the 22Na efflux, suggesting that the transitory nature of these effects is due to stimulation of uninhibited Na-K pumps. The transient effects of low ouabain concentrations contrasted with binding experiments where, with 1 X 10(-6) M-ouabain, the binding first peaked after 30 min with only a small decline thereafter. With higher concentrations of ouabain (1 X 10(-4) and 1 X 10(-3) M), the potentiating effect was also transitory, but 22Na efflux was permanently inhibited and the [Na]i rose. The possibility of there being two populations of ouabain binding sites having different affinities, was supported by the observation of shoulders in the ouabain concentration-effect characteristics obtained with respect to the mechanical potentiation of noradrenaline responses, the 22Na efflux and the acute ouabain binding. In all cases a shoulder was seen between 1 X 10(-6) and 1 X 10(-5) M-ouabain. The results are consistent with the vasoconstrictor effect of ouabain being due to depolarization of the membrane as a consequence of inhibition of the Na-K pump.