The fatty acid composition of phospholipids plays a key role in the structural and functional properties of cellular membrane. In this study, it was hypothesized that conjugated linoleic acid (CLA) isomer supplementation changes the unsaturation degree of the fatty acids of neutral lipids (NLs) but not those of polar lipids (PLs). Thus, the main goal was to determine the pattern of fatty acid incorporation into hepatic PL and NL fractions. Wistar male rats were fed cis(c)9,trans(t)11 and t10,c12 CLA isomers, separately or as a mixture. Whereas the t10,c12 isomer incorporation in the PL fraction was similar when supplemented either individually or as a mixture, the c9,t11 isomer reached the highest values of incorporation when combined with t10,c12. In the PL fraction, the linoleic acid did not change; but the arachidonic acid decreased, especially in the rats given the mixture. Also in this fraction, the t10,c12 isomer, either separately or as a mixture, decreased the amounts of n-6 long-chain (LC) polyunsaturated fatty acids (PUFA) and increased those of the n-3 LC PUFA relative to the control. In the NL fraction, linoleic acid incorporation followed the diet composition, whereas the arachidonic acid was similar among treatments. Facing CLA isomer supplementation, the present study suggests that fatty acid incorporation into phospholipids, through the balance between n-6 and n-3 LC PUFA, is dependent upon maintaining the unsaturation degree of cellular membrane.