The relationship between the number of3H-ouabain binding sites and the Na, K-pump mediated K-uptake has been characterized in rat soleus muscle. By brief exposure to3H-ouabain (1×10-6-1×10-5mol/l) in vitro, it could be measured that 19-94% of the ouabain binding sites had been occupied. This was associated with a proportionate decrease in the ouabain suppressible K-uptake indicating that under strictly standardized conditions, measurements of3H-ouabain binding sites quantify functional Na,K-pumps. When 3 week old rats were K-depleted for a further week followed by K-repletion 2 h before measurements, the3H-ouabain binding site concentration was 61% lower than in age-matched control soleus muscles. However, the ouabain suppressible K-uptake was only reduced by 35% partly because intracellular Na remained higher in the muscles obtained from K-depleted rats. From the 1st to the 4th week of life, the3H-ouabain binding site concentration increased 2.9-fold. In contrast, the ouabain suppressible K-uptake decreased by a factor 3.5. Accordingly, in muscles from 1 week old rats, the ouabain suppressible K-uptake per3H-ouabain binding site was 10-fold higher than in muscles from 4 week old rats. This difference could not be accounted for by changes in intracellular Na, total or extracellular water. It may be related to differentiation and change in structure. On the basis of the present results and those reported in the literature for mouse and frog skeletal muscle it was calculated that under resting conditions at 30°C in vitro, isolated skeletal muscles only utilize between 3 and 25% of their total capacity for active Na, K-transport. Therefore, variations in the total Na, K-pump capacity may not readily be detected in measurements of the ouabain suppressible rate of K-uptake.