[en] Accurate estimation of the specific binding of [³H]ouabain in intact tissues is difficult. Toxic actions of non-labeled ouabain at high concentrations is necessary to prevent specific binding of [³H]ouabain to Na,K-ATPase caused a loss of cell viability and non-specific binding to be underestimated in these preparations. Dissociation of [³H]ouabain from guinea-pig myocytes over 180 min occurred in two phases. The fast-releasing component, which had a half-life of approximately 8 min, accounted for greater than 85% of all [³H]ouabain bound to myocytes following a 60-min incubation in the presence of 50 nM [³H]ouabain. [³H]ouabain bound to the slow releasing site increased with incubation time and with increasing [³H]ouabain concentration. Analysis of [³H]ouabain binding to the fast releasing site yielded non-linear Scatchard plots. Non-linearity appears to result from reduced [³H]ouabain binding due to low intracellular Na⁺ concentration, especially at low [³H]ouabain concentrations. Addition of 2 μM monesin, a Na⁺ ionophore, promoted binding of [³H]ouabain in a manner that Scatchard plots became linear. These results indicate that the fast releasing component of [³H]ouabain binding to guinea-pig myocytes represents a single binding site and that at high concentrations [³H]ouabain promotes its binding to this site by elevating intracellular Na⁺