The goal of the present study was to determine whether endogenous synthesis of n-3 polyunsaturated fatty acids (PUFA) in the fat-1 mouse is comparable to fish oil feeding with respect to kidney n-3 PUFA composition and eicosanoid levels. Wild-type and heterozygous fat-1 mice, capable of synthesizing n-3 PUFA endogenously, were given diets enriched in either n-3 or n-6 PUFA in a 2×2 factorial design and terminated after 12 weeks. Kidney phospholipid fatty acids were analysed by gas chromatography. Kidney eicosanoids were analysed by liquid chromatography tandem mass spectrometry. Relative to control mice fed n-6 PUFA, n-3 PUFA fed and fat-1 mice had higher levels of kidney phospholipid n-3 PUFA, and lower levels of n-6 PUFA and eicosanoids. However, mice fed n-3 PUFA mice had higher levels of n-3 PUFA and lower levels of eicosanoids as compared to fat-1 mice. In conclusion, diet feeding had a greater impact on kidney fatty acid composition and eicosanoid levels than the genetic effect of the fat-1 gene. However, the fat-1 mouse remains a close approximation that can be used as a complementary model to study the role of n-3 PUFA in the kidney.