Biopsies were obtained from the gastrocnemius muscle of 13 male and 12 female distance runners and analyzed for [14C]palmitoyl-CoA oxidation, fiber composition and the activities of selected enzymes. The male and female runners were similar in terms of maximal O2 uptake (.ovrhdot.VO2 max [maximal oxygen consumption]), training mileage, fiber compositions and data collected during a 60 min treadmill run at 70% .ovrhdot.VO2 max. Muscle succinate dehydrogenase and carnitine palmitoyl transferase activities were significantly greater (P < 0.05) in the male than in the female runners. The male runners'' muscle also showed a greater capacity to oxidize palmitoyl CoA. Little relationship was found between muscle lipid metabolism, enzyme activities and the calculated (respiratory exchange) fraction of energy derived from fat during 60 min of running at 70% .ovrhdot.VO2 max. Although endurance training (80-115 km/wk) markedly enhances the capacity of muscles to metabolize fats, the factors that regulate the usage of lipids during prolonged exercise do not appear to be limited by the capacity of the fibers to oxidize fatty acids, as determined by in vitro measurements.