The present study examined the relationships between changes in intra- and extracellular concentrations of strong ions, the appearance of nonvolatile acid (NVA) in venous perfusate, and skeletal muscle fatigue during intense electrical stimulation. A one-pass system was used to perfuse an isolated rat hindlimb during 5 min of intermittent tetanic contractions. Initial isometric tensions averaged 2.85 kg/hindlimb and declined by 45% during 5 min. During stimulation, intracellular lactate concentration ([La-]i) increased by 2, 13, 15, and 21 meq/l of intracellular fluid in the soleus, plantaris, and red and white gastrocnemius. This was associated with a proportionate decrease in intracellular K+ ([K+]i) and Mg2+([Mg2+]i) concentrations and increased intracellular Na+ ([Na+]i) and Cl-([Cl-]i) concentrations. A stoichiometrically equivalent uptake of Na+ and Cl- from the perfusate peaked at 8.5 mu eq.min-1.g-1 at the end of the 5th min. The increase in plasma [K+] during the last 4 min of stimulation was constant at 0.5 mu eq.min-1.g-1. A significant reduction in intracellular strong ion difference of all muscles contributed directly to an increase in [H+] during stimulation. After the 1st min of stimulation the rate of appearance of NVA in venous perfusate exceeded that of the increase in venous plasma [La-] by 12-fold; this decreased to 2.7-fold at the end of 5 min. La- release and NVA appearance in venous perfusate was maximal at 3.1 and 9.7 mu eq.min-1.g wet wt-1 during the 4th min of stimulation. It is concluded that the changes in the intracellular concentrations of strong ions during intense contractile activity are the primary factors contributing to skeletal muscle fatigue.