The purpose of this study was to investigate the hypothesis that Na+-K+-ATPase activity is reduced in muscle of different fiber composition after a single session of aerobic exercise in rats. In one experiment, untrained female Sprague-Dawley rats (weight 275 ± 21 g; means ± SE; n = 30) were run (Run) on a treadmill at 21 m/min and 8% grade until fatigue, or to a maximum of 2 h, which served as control (Con), or performed an additional 45 min of low-intensity exercise at 10 m/min (Run+). In a second experiment, utilizing rats of similar characteristics (weight 258 ± 18 g; n = 32), Run was followed by passive recovery (Rec). Directly after exercise, rats were anesthetized, and tissue was extracted from Soleus (Sol), red vastus lateralis (RV), white vastus lateralis (WV), and extensor digitorum longus (EDL) and frozen for later analysis. 3- O-methylfluorescein phosphatase activity (3- O-MFPase) was determined as an indicator of Na+-K+-ATPase activity, and glycogen depletion identified recruitment of each muscle during exercise. 3- O-MFPase was decreased ( P < 0.05) at Run+ by an average of 12% from Con in all muscles ( P < 0.05). No difference was found between Con and Run. Glycogen was lower ( P < 0.05) by 65, 57, 44, and 33% (Sol, EDL, RV, and WV, respectively) at Run, and there was no further depletion during the continued low-intensity exercise period. No differences in Na+-K+-ATPase activity was observed between Con and Rec. The results of this study indicate that inactivation of Na+-K+-ATPase can be induced by aerobic exercise in a volume-dependent manner and that the inactivation that occurs is not specific to muscles of different fiber-type composition. Inactivation of Na+-K+-ATPase suggests intrinsic structural modifications by mechanisms that are unclear.