Sphingomyelin has been shown to be a source of bioactive compounds. This sphingolipid is located mostly in the outer layer of the plasma membrane and in the membranes of organelles. Sphingomyelin located in the plasma membrane is hydrolyzed into ceramide and phosphorylcholine. Ceramide is the principal second messenger in the sphingomyelin transmembrane signaling pathway. Products of ceramide metabolism, namely, sphingosine, sphingosine-1-phosphate, and ceramide-1-phosphate, also exert broad biological effects. The major effects of ceramide are induction of differentiation, inhibition of proliferation, regulation of inflammatory processes, and induction of apoptosis. There is also convincing evidence that ceramide counteracts insulin-stimulated glucose uptake. Ceramides are also present in skeletal muscles. We investigated ceramide metabolism in different skeletal muscle types of the rat at rest and after prolonged exercise of moderate intensity. Exercise reduced the total content of ceramide fatty acids and changed their composition in each muscle type. These data indicate that the sphingomyelin-signaling pathway functions in skeletal muscles and that its activity is downregulated during prolonged exercise. The content of ceramide in the muscles was inversely related to 2-deoxyglucose uptake by the muscles. This indicates that ceramide may be involved in regulation of glucose uptake by skeletal muscles in vivo.