We investigated the effect of regular exercise and changes in dietary fatty acid profile on skeletal muscle phospholipid fatty acid profile in rats. Rats were randomly divided into three groups and for 4 wk fed either a carbohydrate-rich diet (CHO, 10 percent of total energy (E%) fat, 20 E% protein, 70 E% CHO) or one of two fat-rich diets (65 E% fat, 20 E% protein, 15 E% CHO) containing predominantly either saturated or monounsaturated fatty acids. Each dietary group was randomly assigned to a trained (6 d/wk, progressive to 60 min, 28 m/min at a 10 degrees incline) or a sedentary group. The effect of training was apparent in the three hindlimb muscles analyzed: red quadriceps, white quadriceps and soleus. The unsaturation index was significantly lower in the trained than in the sedentary groups (206 +/- 2 vs. 215 +/- 2, P < 0. 01), which largely reflected a lower content of arachidonic acid [20:4(n-6): 14.5 +/- 0.5 vs. 16.6 +/- 0.4% of total fatty acids, P < 0.01] and docosahexaenoic acid [22:6(n-3): 11.1 +/- 0.2 vs. 11.7 +/- 0.3% of total fatty acids, P < 0.03] and a concomitant higher content of linoleic acid [18:2(n-6): 20.0 +/- 0.4 vs. 17.8 +/- 0.4% of total fatty acids, P < 0.01]. Training affected skeletal muscle membrane structural composition, and this occurred independently of dietary fatty acid changes. This change likely reflects an increased utilization of highly unsaturated fatty acids for energy, an effect which may have deleterious effects on insulin action.