The synthesis of n-3 and n-6 polyunsaturated acids (PUFAs) is associated with physiological functions in mammals, being catalyzed by Δ-5D and Δ-6D desaturases and elongases Elovl-2 and Elovl-5. In this context, we aimed to study the chief kinetic features of PUFA liver anabolism, looking upon (i) the time-dependency for the specific activity of Δ-6D, Δ-5D, Elovl2, Elovl2/5 and Elovl5, using n-3 and n-6 precursors between 0 and 240 min ex vivo in mouse liver.; and (ii) the specific activity-substrate (α-linolenic acid; ALA) concentration responses of Δ-6D in the absence and presence of linoleic acid (LA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), an enzyme regarded as the rate-limiting step in PUFA anabolism. Mouse liver was obtained from eight-week-old Balb/c mice fed a chow diet (expressed as % of total calories: 18 % fat, 24 % protein, and 58 % carbohydrate, with a caloric value of 3.1 kcal/g) for eight weeks, and used for preparation of the microsomal fraction. Enzymatic activities assayed under the addition of specific PUFA precursors or LA, ARA, EPA and DHA, identifying the respective PUFA products as fatty acid methyl esters by gas chromatographic analysis. Data described corroborate that (i) PUFA metabolism mainly occurs in the liver, with the participating enzymes preferring n-3 than n-6 substrates; and show that (ii) the rate-limiting step of PUFA metabolism relies on the second reaction of Δ-6D (24:5n-3 transformed to 24:6n-3); and (iii) LA, ARA, EPA and DHA act as non-competitive inhibitors with respect to ALA in the reaction catalyzed by Δ-6D. These results are relevant for future studies concerning the metabolic and nutritional implications of changes in desaturation and elongation of PUFAs.