Uptake of polyunsaturated fatty acids by the rat brain occurs mainly during the three weeks before weaning. Docosahexaenoic acid [22:6], the predominant polyunsaturated fatty acid in the adult brain, appears to be preferentially taken up from the circulation by both the adult and developing rat brain. To test the hypothesis that this preferential incorporation was mediated by the cerebral microvasculature, we compared the incorporation of 22:6 to a saturated fatty acid, palmitic acid [16:0], in freshly isolated rat brain microvessels from the pooled brains of entire litters of two-week-old rats (n=8 litters). For each litter duplicate incubations with 2 muCi of [1-C-14]22:6 or [1-C-14]16:0 were performed in 60% autologous rat serum for 2 hr at 37-degrees-C. [H-3]Sucrose was included in each incubation, allowing correction for non-specific uptake and trapping. An average of 2.7 +/- 2.0% (SD) of the radioactivity from 16:0 was found in the microvessels after 2 hr, vs 0.9 +/- 0.6% for 22:6. This yielded a three-fold enrichment of 16:0 over 22:6 (P=0.02, paired t-test). There was preferential incorporation of 22:6 into phosphatidylethanolamine and of 16:0 into phosphatidylcholine, although most of the label from either substrate remained as fatty acid after the 2 hr incubation. These results do not indicate that brain capillaries mediate the preferential incorporation of polyunsaturated fatty acids into brain tissue that was seen in intact young rats.