In rat skeletal muscle prolonged exercise affects the content and composition of ceramides, but in human skeletal muscle no data are available on this compound. Our aim was to examine the content of ceramide- and sphingomyelin fatty acids and neutral, Mg(2+)-dependent sphingomyelinase activity in skeletal muscle in untrained and trained subjects before and after prolonged exercise. Healthy male subjects were recruited into an untrained (n = 8, VO2,max 3.8 +/- 0.2 1 min1) and a trained (n = 8, Vo2,max 5.1 +/- 0.1 1 min2) group. Before and after a 3-h exercise bout (58 +/- 1% VO2,max) a muscle biopsy was excised from the vastus lateralis. Ceramide and sphingomyelin were isolated using thin-layer chromatography. The content of individual ceramide fatty acids and sphingomyelin fatty acids was measured by means of gas-liquid chromatography. The activity of neutral, Mg(2+)-dependent sphingomyelinase was measured using N-[14CH3]-sphingomyelin as a substrate. Prior to exercise, the muscle total ceramide fatty acid content in both groups was similar (201 +/- 18 and 197 +/- 9 nmol g(-1) in the untrained and trained group, respectively) and after exercise a 25% increase in the content was observed in each group. At rest, the muscle total sphingomyelin fatty acid content was higher in untrained than in trained subjects (456 +/- 10, 407 +/- 7 nmol g(-1), respectively; P < 0.05). After exercise a 20% increase (P < 0.05) in total sphingomyelin was observed only in the trained subjects. The muscle neutral, Mg(2+)-dependent sphingomyelinase activity was similar in the two groups at rest and a similar reduction was observed after exercise in both groups (untrained from 2.19 +/- 0.08 to 1.78 +/- 0.08 and trained from 2.31 +/- 0.12 to 1.80 +/- 0.09 nmol (mg protein) (-1) h(-1); P < 0.05 in each case). In conclusion, we have reported, for the first time, the values for ceramide fatty acid content and neutral, Mg2(+)-dependent sphingomyelinase activity in human skeletal muscle. The results indicate that acute prolonged exercise affects ceramide metabolism in human skeletal muscle both in untrained and in trained subjects and this may influence muscle cell adaptation and metabolism.