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 alpha- and beta-subunits were quantified by Western blotting. Exercise significantly increased (P < 0.05) the vesicular membrane content of the alpha(2)-, total alpha-, and beta(1)-subunits by 70 +/- 29, 35 +/- 10, and 26 +/- 5%, respectively. The membrane content of alpha(1) 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 alpha(2)-, total alpha-, and beta(1)-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.