Six human subjects performed one-legged knee extensor exercise (90 ± 4 W) until fatigue (exercise time 4.6 ± 0.8 min). Needle biopsies were obtained from vastus lateralis muscle before and immediately after exercise. Production of giant sarcolemmal vesicles from the biopsy material was used as a membrane purification procedure, and Na⁺-K⁺ pump α-and β-subunits were quantified by Western blotting. Exercise significantly increased (P < 0.05) the vesicular membrane content of the α₂-, total α-, and β₁-subunits by 70 ± 29, 35 ± 10, and 26 ± 5%, respectively. The membrane content of α₁ was not changed by exercise, and the densities of subunits in muscle homogenates were unchanged. The ratio of vesicular to crude muscle homogenate content of the α₂-, total α-, and β₁-subunits was elevated during exercise by 67 ± 33 (P < 0.05), 23 ± 6 (P < 0.05), and 40 ± 14% (P = 0.06), respectively. It is concluded that translocation of subunits is an important mechanism involved in the short time upregulation of the Na⁺-K⁺ pumps in association with human muscle activity.