The sphingomyelin-signaling pathway has been described in many tissues. Ceramide is the main second messenger in this pathway. Ceramide has also been shown to be present in skeletal muscles; however, there are few data on the regulation of the content of ceramide in muscle tissue. Moreover, there are no data on the content of particular ceramides or their composition in the muscles. The aim of the present study was to examine the content and composition of fatty acids (FA) in ceramide and sphingomyelin moieties and the activity of neutral Mg(2+)-dependent sphingomyelinase in different skeletal muscle types of the rat at rest and after exhausting exercise of moderate intensity. The experiments were carried out on male Wistar rats, divided into two groups: 1) control and 2) exercised until exhaustion on a treadmill. Soleus and red and white gastrocnemius muscles were taken. Ceramide and sphingomyelin were separated by TLC. The content of individual FA in the two compounds was determined by gas-liquid chromatography. Twelve different ceramides and sphingomyelins were identified and quantified in each muscle type according to the FA residues. Saturated FA consisted of 68, 67, and 66% of total ceramide-FA and 75, 77, and 78% of total sphingomyelin-FA in soleus and red and white gastrocnemius, respectively. The total content of ceramide- and sphingomyelin-FA and the activity of sphingomyelinase were highest in the soleus and lowest in the white gastrocnemius. Exercise resulted in a reduction in the total content of ceramide- and sphingomyelin-FA in each muscle. This was accounted for mostly by a reduction in content in the amount of saturated FA. Exercise reduced the activity of neutral Mg(2+)-dependent sphingomyelinase in the soleus and red gastrocnemius and did not affect its activity in the white gastrocnemius. We conclude that the sphingomyelin-signaling pathway is present in skeletal muscles and that it is influenced by prolonged exercise.