Exercise has been reported to increase the Na+-K+-ATPase (Na+-K+ pump) alpha2 isoform in the plasma membrane 1.2- to 1.9-fold, purportedly reflecting Na+-K+ pump translocation from an undefined intracellular pool. We examined whether Na+-K+ pump stimulation, elicited by muscle contraction or insulin, increases the plasma membrane Na+-K+ pump content ([3H]ouabain binding) in muscles from young rats. Stimulation of isolated soleus muscle for 10 s at 120 Hz caused a rapid rise in intracellular Na+ content, followed by an 18-fold increase in the Na+ re-extrusion rate (80 % of theoretical maximum). Muscles frozen immediately or 120 s after 10-120 s stimulation showed 10-22 % decrease in [3H]ouabain binding expressed per gram wet weight, but with no significant change expressed per gram dry weight. In soleus muscles from adult rats, [3H]ouabain binding was unaltered after 10 s stimulation at 120 Hz. Extensor digitorum longus (EDL) muscles stimulated for 10-60 s at 120 Hz showed no significant change in [3H]ouabain binding. Insulin (100 mU ml-1) decreased intracellular Na+ content by 27 % and increased 86Rb uptake by 23 % soleus muscles, but [3H]ouabain binding was unchanged. After stimulation for 30 s at 60 Hz soleus muscle showed a 30% decrease in intracellular Na+ content, demonstrating increased Na+-K+ pump activity, but [3H]ouabain binding measured 5 to 120 min after stimulation was unchanged. Stimulation of soleus or EDL muscles for 120-240 min at 1 Hz (continuously) or 10 Hz (intermittently) produced no change in [3H]ouabain binding per gram dry weight. In conclusion, the stimulating effects of electrical stimulation or insulin on active Na+, K+-transport in rat skeletal muscle could not be even partially accounted for by an acute increase in the content of functional Na+ -K+ pumps in the plasma membrane.