We studied the effects of a diet that was low in fat, high in carbohydrate (CHO) on milk lipid composition and de novo endogenous fatty acid synthesis by the mammary gland in five lactating women. The women consumed either a low fat (LF) (5% fat, 80% CHO) diet or a high fat (HF) (40% fat, 45% CHO) diet. Fat synthesis was determined after an oral dose of 500 mg/kg D2O by measuring the incorporation of deuterium into C10:0 to C18:0 saturated fatty acids of milk fat and plasma triglycerides by gas chromatography-mass spectrometry. Synthesis of plasma C16:0 and C18:0 triglycerides was barely detectable while women consumed the HF diet, but was increased 6-fold during the LF diet. Medium chain fatty acids secreted by the mammary gland increased from 12.8% (HF diet) to 16.3% (LF diet) in milk fat from four of five subjects (p = 0.027). Medium chain fatty acid secretion, however, increased from 13.9% (HF diet) to 29.9% (LF diet) in one subject. The primary fatty acids synthesized during lactation were C10:0, C12:0, and C14:0 in the majority of women studied. The LF diet significantly increased the apparent synthesis of C14:0 (p = 0.05), whereas no changes were observed in C12:0, C16:0, or C18:0. One subject had highly enriched C16:0 and C18:0 fatty acids in her milk on the LF diet, which could have been the result of mammary synthesis or of transport and secretion of hepatically synthesized lipids.