Human studies and some animal work have shown more docosahexaenoic acid (DHA) and arachidonic acid (ARA) was accumulated or converted from precursors in females compared to males. This study explored in-depth the effect of gender on fatty acid composition and polyunsaturated fatty acid metabolism in rats fed one of two well-defined diets containing 10% total fat. One diet contained 15% of linoleic acid (LA) and 3% of α-linolenic acid (ALA) of the total fatty acids (LA+ALA diet), while the other diet contained 15% LA and 0.05% ALA (LA diet). At the age of 20 weeks, all animals were orally administered a single dose of a mixture of deuterium-labeled LA and ALA. Caudal venous blood was then drawn at 0, 2, 4, 8, 12, 24, 48, 96 and 168 h. The concentrations of the deuterated precursors and their metabolites in plasma total lipids were quantified by GC/MS negative chemical ionization. Endogenous fatty acids were quantified by GC/FID analysis. When expressed as the percentage of oral dosage, female rats accumulated more precursors and more products, deuterated DHA and deuterated n-6 docosapentaenoic acid (2H5-DPAn-6), in plasma than did male rats in both the LA+ALA diet and the LA diet. For the endogenous non-labeled PUFA, greater concentrations of DHA and DPAn-6 were similarly observed in female rats compared to males within each diet. A lower concentration of non-labeled ARA was observed only in female rats fed the LA+ALA diet. In summary, greater endogenous and exogenous DHA and DPAn-6 was observed in female rat plasma and this was independent of dietary ALA status.