The concentration and fatty acid composition of phospholipids in animal cells are important determinants of membrane function. Membrane function may influence apoptosis, a biological process that is crucial for the normal development and function of the body. Few and conflicting data exist regarding the effect of chronic exercise on apoptosis in skeletal muscle, and no data exist regarding the effect of chronic exercise on the fatty acid composition of individual muscle phospholipids. We therefore examined the effects of 8 weeks of voluntary wheel running on DNA fragmentation (an index of apoptosis) and on the concentration and fatty acid composition of individual muscle phospholipids and ceramide (a lipid involved in apoptotic signalling) in rat gastrocnemius muscle by comparing 11 trained and 14 untrained male Wistar rats. The trained animals had significantly (P < 0.05) higher cytochrome c oxidase activity (an index of aerobic adaptation) and lower phosphatidyl inositol concentration compared with their untrained counterparts. Groups did not differ in DNA fragmentation or any other lipid parameter. Our findings suggest that chronic wheel running did not affect apoptosis or the concentration and fatty acid composition of most phospholipids and ceramide in rat gastrocnemius muscle. Given the participation of several phospholipids and ceramide in apoptotic signalling, it appears that the lack of changes in the lipid parameters is in agreement with the lack of change in DNA fragmentation with exercise.