Abstract: Docosahexaenoate is important for normal neural development. It can be derived from α-linolenate, but carbon from α-linolenate is also recycled into de novo lipid synthesis. The objective of this study was to quantify the amount of α-linolenate used to produce docosahexaenoate versus lipids synthesized de novo that accumulate in the brain of the developing rat. A physiological dose of carbon-13-labeled α-linolenate was injected into the stomachs of mother-reared 6-day-old rat pups. Total lipids of brain, liver, and gut were extracted from rats killed 3 h to 30 days after dosing. Carbon-13 enrichment was determined by isotope ratio mass spectrometry. Carbon-13-enriched α-linolenate was not detected in the brain at any time point, and its levels in liver and gut exceeded detection limits at most time points, so tracer mass was quantified mainly for three end products—docosahexaenoate, palmitate, and cholesterol. Carbon-13-enriched cholesterol, palmitate, docosahexaenoate, and water-soluble metabolites were detected in brain, liver, and gut. Enrichment (in micrograms of carbon-13 per organ) in brain cholesterol exceeded that in brain docosahexaenoate by four- to 16-fold over the duration of the study. Enrichment in brain palmitate exceeded that in brain docosahexaenoate by three- to 30-fold over the first 8 days of the study. These results indicate that carbon from α-linolenate is not exclusively conserved for synthesis of longer n-3 polyunsaturates but is a readily accessible carbon source for de novo lipogenesis during early brain development in the suckling rat. Owing to a high rate of β-oxidation and carbon recycling, dependence on α-linolenate as the sole source of docosahexaenoate may incur a potential risk of providing insufficient docosahexaenoate for the developing brain.