Previous studies have shown that chronic administration of SR-3 (a 1:4 mixture of alpha-linolenic and linoleic acid) affects spatial learning, thermoregulation, pain threshold, and protection from seizures. The mode of action of SR-3 is unknown. One possible explanation is that SR-3 induces changes in the FA profile and in the cholesterol level in neuronal membranes. This study used 10 independent groups of rats (ni = 12) given 4 weeks of either saline, mineral oil (vehicle), alpha-tocopherol (antioxidant), alpha-linolenic acid, linoleic acid, or one of 5 different ratios of alpha-linolenic acid:linoleic acid (1:3, 1:4, 1:5, 1:6, 1:7) as free fatty acids. FA profile and cholesterol level were examined by GC method in synaptosomes obtained from the frontal cortex of the rats. The mineral oil treated group served as the control group. No difference was found in the FA profile or cholesterol level except for the SR-3 treated group. The ratio of 1:4 was found to have a significant influence on decreasing the cholesterol level and in inducing major changes in the FA profile, such as an increase in EFA. These effects of SR-3 may result in modification of the membrane fluidity, which may, in turn, enhance cognitive and neuropharmacological effects.