The influence of age on the binding of3H-ouabain in skeletal muscle has been characterized in rats, mice and guinea pigs. Measurements performed using biopsies and intact fibers obtained from different types of rat muscles showed that from birth to the 4th week of life, the number of3H-ouabain binding sites per unit weight increases up to 5-fold, followed by almost the same relative decrease to a plateau around 250 pmol/g wet wt at an age of 22 weeks. These changes were not associated with any major alterations in apparent KD (1.7-3.1×10-7M) dissociation rate or heterogeneity in binding characteristics. Measurements of 3-O-methylfluorescein phosphatase activity, an enzyme activity which is closely correlated to the Na-K-ATPase activity, confirmed the3H-ouabain binding data. In mice, the number of3H-ouabain binding sites showed similar, albeit less pronounced changes with age, a maximum being reached at the 4th week of life. In guinea pigs, the number of3H-ouabain binding sites per unit weight decreased by 60% from birth to maturity. The results indicate that the early development and differentiation of individual skeletal muscles is associated with a marked increase in the number of Na-K-pumps (when expressed as pmol/muscle), until at maturity a plateau is reached. However, when expressed as pmol/g wet wt the increase is followed by a decrease to a plateau. This may in part account for the relatively low digitalis sensitivity seen in infants as compared to newborn and mature individuals.