1. Following intraperitoneal injection of [3H]ouabain in rats, the isotope is rapidly distributed in blood plasma available for binding to the Na‐K‐ATPase in the plasma membranes of most tissues. In skeletal muscle tissue excised and washed 4 × 30 min in ice‐cold buffer, 95% of the 3H activity retained was shown to be [3H]ouabain using a specific binding assay. 2. The [3H]ouabain bound to soleus and extensor digitorum longus (e.d.l.) muscles in vivo and retained following wash‐out in the cold showed the same saturation characteristics as those determined when binding took place in vitro. 3. In soleus and e.d.l. muscles obtained from 28‐day‐old rats, the number of [3H]ouabain binding sites measured in vivo was 583±19 and 720±22 pmol/g wet wt., respectively, i.e. in good agreement with previous and present results obtained in vitro. 4. In vivo measurements showed that 7 days after denervation, the number of [3H]ouabain binding sites in soleus and e.d.l. muscles was reduced by 22 and 13%, respectively. 5. In the age interval from 28 to 85 days, the number of [3H]ouabain binding sites in soleus was found to decrease by 58%. Following I.P. injection of [3H]ouabain, the 85‐day‐old rats showed a more pronounced and sustained rise in plasma 3H activity, which in part can be due to the reduced capacity for [3H]ouabain binding in skeletal muscle. 6. K depletion induced by the administration of K‐deficient diet for 3 weeks reduced [3H]ouabain binding by 63% in soleus muscles. In the K‐depleted animals, the plasma 3H activity measured 15 min after I.P. injection of [3H]ouabain was 77% higher than in controls receiving the same dose per kg body weight. 7. The present in vivo results provide further support for the idea that increased digitalis toxicity due to increasing age or K depletion is related to reduced binding capacity for digitalis glycosides in skeletal muscle.