K-depletion induced by K-deficient fodder led to hypokalemia, decreased K-content and increased Na-content in skeletal muscle of rats. The heart showed similar, although more modest changes, whereas brain, erythrocytes and liver maintained virtually constant Na-K-contents. K-depletion decreased total binding capacity for 3H-ouabain by up to 76%, an effect which could be demonstrated both in vitro and in vivo. Following 3 weeks of K-depletion, the apparent KD for 3H-ouabain binding to rat soleus was 1.3 X 10(-7) M as compared to 2.4 X 10(-7) M in controls. Also in mice and guinea pigs, K-depletion induced a selective loss of K from muscle and decreased 3H-ouabain binding capacity. K-depletion induced by diuretics or fluorohydrocortisone gave similar effects. The effects of K-depletion on 3H-ouabain binding capacity were confirmed by measurements of 3-O-methylfluorescein phosphatase activity, an enzyme activity which is closely correlated to the Na-K-ATPase activity. Following readministration of K, the K-contents of plasma and muscle reached control levels in 24 hours, but 3H-ouabain binding capacity was not normalized until after 6 days of K-repletion. In mammalian skeletal muscle, K-depletion leads to a marked and reversible reduction in 3H-ouabain binding capacity, which may be secondary to the selective loss of K or gain of Na.