The purpose of this experiment was to study endurance performance and substrate storage and utilization in fat- or carbohydrate-fed rats. Ninety-nine rats were randomly divided into three groups and over 4 wk were fed either a carbohydrate-rich [CHO; 10% total energy content in the diet (E%) fat, 20 E% protein, 70 E% carbohydrate] diet or one of two fat-rich diets (65 E% fat, 20 E% protein, 15 E% carbohydrate) containing either saturated (Sat) or monounsaturated fatty acids (Mono). Each dietary group was randomly assigned to a trained (6 days/wk, progressive to 60 min, 28 m/min at a 10% incline) or a sedentary group. Rats were killed either before or after a treadmill endurance run to exhaustion. Training increased endurance (206%), but diet composition did not affect endurance in either trained or sedentary rats. -Hydroxyacyl-CoA dehydrogenase activity was increased in fat-fed but not carbohydrate-fed rats (P < 0.05). Respiratory exchange ratio during the initial phase of exercise was lower after the Mono compared with the Sat diet (P < 0.05) and higher after the CHO than the Sat diet (P < 0.05). Thus adaptation to a high-fat diet containing a moderate amount of carbohydrates did not induce enhanced endurance in either trained or untrained rats; however, substrate utilization was modulated by both amount and type of dietary fat during the initial stage of exercise in trained and sedentary rats.