Little is known about the long-term effects of diet restriction, exercise, and aging on the fatty acid composition of adipose tissue. To investigate these effects, male Wistar rats were placed in one of four experimental groups: 1) ad libitum fed, nonexercised; 2) ad libitum fed, exercised; 3) diet restriction, nonexercised; and 4) diet restriction, exercised. Exercise consisted of swimming on alternate days, and diet-restricted animals were fed every other day. Animals were assigned to one of the four treatment groups at weaning, and the fatty acid composition in the epididymal, perirenal, and inguinal fat depots were determined every 4 months from 12 to 28 months of age. All animals were fed the same diet so the relative percentage of fatty acids in the diet did not contribute to the changes observed in adipose tissue fatty acid composition. Regardless of treatment groups and fat depot, the concentration of saturated fatty acids in adipose tissue tended to decrease as body weight increased. Saturated fatty acid composition was greater in the diet-restricted than the ad libitum-fed groups. The saturated fatty acid concentration was different between the three fat depots (perirenal 27.8%, inguinal 24.7%, and epididymal 21.6%; P < 0.001). The linoleic acid content did not change with age in the epididymal or perirenal fat depot; however, the change was significant for the inguinal fat depot. Following a large decrease in inguinal linoleic concentration through 20 months, linoleic acid concentration increased in all treatments between 20 and 28 months. The results indicate that diet restriction, exercise, and aging have significant effects on adipose tissue fatty acid composition and these effects are different depending on the anatomical location of the adipose tissue depot.