The effect of dietary restriction of n-3 fatty acids during development on brain phospholipid fatty acid composition and exploratory behavior has been studied in male Sprague Dawley rats. Female rats were fed semipurified diets containing either 5.5% safflower oil or 6% soybean oil for 6 wk prior to mating and throughout gestation and lactation. Control rats were maintained on laboratory chow. The male pups were weaned to the diets of the dams except for one group which was switched from safflower to soybean oil at weaning. Behavioral studies and brain phospholipid analyses were conducted at 16-18 wk of age. Rats fed safflower oil showed significantly lower levels of 22:6n-3 in phospholipids of synaptic membranes and myelin than rats fed soybean oil or chow. The decrease in 22:6n-3 was compensated for by an increase in 22:5n-6, the total content of polyunsaturated fatty acids remaining approximately constant. The brain phospholipid fatty acid composition of rats switched from safflower to soybean oil at weaning was similar to that of rats fed soybean oil throughout the experiment. There was no difference in spontaneous locomotor activity among the different dietary groups. However, rats raised on safflower oil displayed a significantly lower exploratory activity (horizontal movements and rearings) in a novel environment than rats fed soybean oil or chow. In contrast to the brain phospholipid fatty acid composition, there was no recovery of exploratory behavior in rats raised on safflower oil and switched to soybean oil at weaning suggesting a specific requirement of n-3 fatty acids during development.