A strenuous program of treadmill running increased endogenous lipoprotein lipase (LPL) activity 2.7-fold in the soleus and 2.8-fold in fast-red fibers of the quadriceps. Under these conditions, intracellular triacylglycerols (TG) decreased 25% in the soleus and 44% in fast-red muscle fibers. Free fatty acids (FFA) increased 2.1-fold (soleus) and 2.7-fold (fast-red). In the heart, a 2-h bout of swimming in untrained rats increased intracellular LPL activity 64% measured 24 h after work. The magnitude of the increase in endogenous LPL activity in heart was dependent on the intensity of the exercise. With elevated levels of intracellular LPL activity in the myocardium, TG was reduced 49% and FFA increased 3.4-fold. These results provide evidence that exercise may result in an increased capacity to hydrolyze intramuscular TG. Further, they suggest that LPL may be the enzyme responsible for intracellular TG hydrolysis in heart and skeletal muscle.