Docosahexaenoic acid (DHA), a 22-carbon, highly unsaturated, n-3 fatty acid, is important for optimal nervous system function. In this study, designed to quantify how preformed dietary DHA regulates metabolic pathways in vivo, 8-d-old rat pups were divided into four groups and fed artificial rat milk diets. One group was fed formula with deuterium-labeled LNA (d5-LNA) as the only source of n-3 fatty acids, and a second group was fed formula that contained d5-LNA and unlabeled DHA. Two additional groups were dam-reared to permit analysis of fatty acyl pool sizes at postnatal days 8 and 28. The dams were fed a diet that contained 3% unlabeled LNA. DHA in brain and liver was analyzed. Our study demonstrated that preformed DHA in the diet markedly decreased the amount of biosynthesized DHA that accumulated in the brain and the liver. Surprisingly, 40% of the DHA that was newly acquired during this period in the "LNA" group was unlabeled. Because there were no unlabeled n-3 fatty acids in this diet, this DHA must have been derived from body stores of n-3 fatty acids. Thus, body stores can be a significant source of brain DHA in animals that are fed LNA as the only source of n-3 fatty acids. Abbreviations AA, arachidonic acid DHA, docosahexaenoic acid d5-DHA, deuterium-labeled docosahexaenoic acid d5-LNA, deuterium labeled ␣-linolenic acid DPAn-6, docosapentaenoic acid EE, ethyl ester EPA, eicosapentaenoic acid GC, gas chromatography LCP, long-chain polyunsaturate LNA, ␣-linolenic acid MS, mass spectrometry Docosahexaenoic acid (DHA) is a 22-carbon, highly unsaturated, n-3 fatty acid that accumulates in high concentrations in the brain and the retina. It is known that DHA is important for optimal nervous system function as significant deficiency states are associated with decreased behavioral function in animal models (1-8). N-3 fatty acids are essential nutrients for mammals, that is, they cannot be synthesized de novo. However, longer chain n-3 fatty acids such as DHA can be biosynthesized in mammals from n-3 fatty acid precursors such as the 18-carbon n-3 fatty acid ␣-linolenic acid (LNA) (9 -13).An important question for essential fatty acid nutrition has been whether preformed dietary DHA is required or needs can be met with its precursor LNA. Although breast milk always contains DHA (14), until 2002, no infant formula that contained DHA was available in North America as n-3 fatty acids were supplied only in the form of LNA.Whether it is reasonable to expect metabolism of LNA to supply all of the DHA needed for brain and other organ growth remains unclear. Data from previous tracer studies show a marked neural tissue preference for preformed DHA over metabolized LNA during development (15,16). Furthermore, animals that ingest diets with high levels of LNA are unable to reach brain DHA levels of animals that ingest .This study therefore was designed to quantify the amount of brain and liver DHA derived from biosynthesis when preformed DHA was or was not available in the diet. It has been suggested that liver micro...