Diets rich in fats produce large quantities of milk with high lipid concentrations, which may be important for the growth of neonates. The present study investigates the effect of different fat enriched diets on mammary gland lipogenesis in lactating rats. Rats were fed for 6 weeks during pregnancy through to mid-lactation with diets containing chow supplemented with 20% (w/w) coconut oil, olive oil or corn oil. The control animals were fed with a low fat (0.5%, w/w) diet, or with chow (6.8%, w/w fat). Rats fed either the 20% (w/w) olive oil-supplemented diet or the 20% (w/w) corn oil-supplemented diet produced milk with significantly lower total fat concentrations (p<0.05) than rats fed the low-fat (0.5% (w/w) corn oil) control diet or with chow (6.8% (w/w) fat. Rats on the olive oil-and corn oil-supplemented diets produced milk that had significantly lower concentrations of total fat and of C8:0 -C18:0 fatty acids and higher concentrations of C18:1 -C18:3 acids compared to the low-fat diet, chow, or the coconut oil-supplemented diets. Compared with the low-fat control diet, all the other dietary regimes suppressed overall fatty acid synthesis in both the lactating mammary gland and liver, with the highest suppression being produced by the olive oil-and corn oil-supplemented diets on mammary fatty acid synthesis. Measurements of the total activity in the mammary gland of the rate-limiting enzyme, acetyl-CoA carboxylase, and of the proportions of the polymeric (active) and protomeric (inactive) forms of this enzyme, showed that the total activity decreased in parallel with the overall rate of fatty acid synthesis. This trend was, however, not noticed for fatty acid synthetase (another lipogenic enzyme). By contrast, a constant proportion of polymeric to protomeric forms was maintained at mid-lactation irrespective of the diet, indicating a possible role of prolactin in mammary gland acetyl-CoA carboxylase polymerization. This study, therefore, showed that the fatty acid composition of the diet as well as hormones involved in lactation may affect mammary gland and liver fatty acid synthesis, through a modification of the rate limiting enzyme of fatty acid synthesis -acetyl-CoA carboxylase.