Muscle contraction may up-regulate the number of Na⁺-K⁺ pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na⁺-K⁺ pump a₂ subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the a₂ subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na⁺-K⁺ pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the a₁ subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the a₂, the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the a subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.