Background: Conjugated linoleic acids (CLA) in general, and in particular the trans-10, cis-12 (t10, c12-CLA) isomer are potent modulators of milk fat synthesis in dairy cows. Studies in rodents, such as mice, have revealed that t10, c12-CLA is responsible for hepatic lipodystrophy and decreased adipose tissue with subsequent changes in the fatty acid distribution. The present study aimed to investigate the fatty acid distribution of lipids in several body tissues compared to their distribution in milk fat in early lactating cows in response to CLA treatment. Effects in mammary gland are further analyzed at gene expression level. Methods: Twenty-five Holstein heifers were fed a diet supplemented with (CLA groups) or without (CON groups) a rumen-protected CLA supplement that provided 6 g/d of c9, t11-and t10, c12-CLA. Five groups of randomly assigned cows were analyzed according to experimental design based on feeding and time of slaughter. Cows in the first group received no CLA supplement and were slaughtered one day postpartum (CON0). Milk samples were taken from the remaining cows in CON and CLA groups until slaughter at 42 (period 1) and 105 (period 2) days in milk (DIM). Immediately after slaughter, tissue samples from liver, retroperitoneal fat, mammary gland and M. longissimus (13th rib) were obtained and analyzed for fatty acid distribution. Relevant genes involved in lipid metabolism of the mammary gland were analyzed using a custom-made microarray platform. Results: Both supplemented CLA isomers increased significantly in milk fat. Furthermore, preformed fatty acids increased at the expense of de novo-synthesized fatty acids. Total and single trans-octadecenoic acids (e. g., t10-18:1 and t11-18:1) also significantly increased. Fatty acid distribution of the mammary gland showed similar changes to those in milk fat, due mainly to residual milk but without affecting gene expression. Liver fatty acids were not altered except for trans-octadecenoic acids, which were increased. Adipose tissue and M. longissimus were only marginally affected by CLA supplementation. Conclusions: Daily supplementation with CLA led to typical alterations usually observed in milk fat depression (reduction of de novo-synthesized fatty acids) but only marginally affected tissue lipids. Gene expression of the mammary gland was not influenced by CLA supplementation.