Conjugated linoleic acid (CLA) has a range of biological properties, including effects on lipid metabolism, milk and body composition in animals. This study investigated the effects of dietary CLA on lactating rats and development of the suckling pups. Dams were fed either a control diet or the same diet supplemented with 25 g/kg of a fat supplement containing 540 g CLA/kg (final concentration of 13·5 g CLA/kg diet) from parturition to the 15th day post-partum. The CLA mixture used in this study contained the following isomers (per 100 g): cis-9, trans-11 (24 g); cis-10, trans-12 (35 g); cis-8, trans-10 (15 g); cis-11, trans-13 (17 g) and others (9 g). On d 15 post partum, CLA supplementation reduced milk fat content by 33% and pup growth by 21%. The milk fatty acid profile, with decreased content of short and medium chain acids, suggests CLA inhibition was more pronounced for de novo lipid synthesis. Consistent with these results, activities of fatty acid synthase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were reduced by CLA treatment in the mammary gland and liver. In contrast, the activity of NADP-malate dehydrogenase was unchanged.