Triacylglycerols make up 98% of the lipid content of milk, ranging in different species from 0 to 50% of the total milk volume. The fatty aid composition of the triacylglycerols depends on the species, the dietary fatty acid composition, and the carbohydrate-to-lipid ratio of the diet. The rate of lipid synthesis in the lactating mammary gland depends on the stage of mammary development and is decreased by fasting and starvation in ruminants and rodents but not in species that fast during lactation, such as seals and hibernating bears. Regulatory agents include insulin, prolactin, and non-esterified fatty acids. Dietary trans fatty acids may depress milk lipid synthesis under certain conditions. Evidence is presented that fatty acids may play a major regulatory role in acute changes in de novo mammary fatty acid synthesis, acting primarily on the activity of acetyl coenzyme A carboxylase.