Using a recently developed method based upon vanadate facilitated3H-ouabain binding, the total concentration of3H-ouabain binding sites was determined in biopsies of rat skeletal muscles containing varying proportions of slow-twitch fibres. In extensor digitorum longus, diaphragm, gastrocnemius and soleus muscles from mature (12-week-old) hyperthyroid rats the values obtained were respectively 2.6, 3.5, 5.1 and 9.8 times higher than those found in the same muscles from hypothyroid animals. This indicates that the effect of thyroid hormones is more pronounced on slow-twitch than on fast-twitch fibres. The changes in3H-ouabain binding site concentration with thyroid status could not be accounted for by differences in affinity or the rate of3H-ouabain binding. In young (4-5 week old) rats, where the K-content and the3H-ouabain binding site concentration in muscle had been reduced by K-depletion, T3-pretreatment produced an even larger relative increase in the3H-ouabain binding site concentration than in age-matched controls, but no increase in K-content. Therefore, the downregulation of3H-ouabain binding sites seen during K-depletion cannot be attributed to a decreased response to thyroid hormones. In normal rats the marked stimulating effect of thyroid hormone on the synthesis of3H-ouabain binding sites was not associated with any significant change in K-content, but clearly preceded by a significant (P<0.001) rise in the efflux of86Rb.