To compare the fatty acid distribution and cholesterol composition in trained and untrained isolated skeletal muscle membranes, a needle biopsy was performed on the vastus lateralis of the 10 distance runners and 10 sedentary men. The muscle sample was homogenized and centrifuged at 100,000 X g; the resulting pellet was analyzed by gas-liquid chromatography for individual fatty acids and cholesterol. The percentage of palmitic acid was significantly lower in the trained muscle tissue. Samples from the distance runners also tended to have a more frequent appearance of linolenic and eicosatrienoic acids, longer fatty acid hydrocarbon chains, and lower cholesterol concentration. It was concluded that trained muscles have an increased membrane fluidity which could beneficially affect the activity of membrane-bound enzymes and active transport. Longer chain length in the membrane lipids may be a means of producing an inner membrane cohesiveness in muscles of trained individuals.