Milk was obtained on postpartum days 2-3 (colostrum) and days 7, 21, 42, and 84 from mothers of 18 very premature (VPT, 26-30 weeks gestational age), 28 premature (PT, 31-36 weeks), and 6 term (T, 37-40 weeks) infants. Lipids were extracted in chloroform-methanol and analyzed by thin-layer (TLC) and gas liquid chromatography (GLC). Fatty acid composition of cholesteryl esters was determined by GLC after isolation of cholesteryl esters by preparative TLC and preparation of methyl esters of the constituent fatty acids. As lactation progressed, amounts of total cholesterol and cholesteryl esters declined. Cholesteryl esters decreased from about 5 mg/dl in colostrum to 1 mg/dl in mature milk. The cholesteryl esters of colostrum from mothers of premature infants were different in fatty acid composition from those of term infants. Proportions of medium-chain saturated fatty acids (12:0, 14:0, 16:0) of preterm colostrum (VPT and PT) were considerably lower than in term colostrum: 23% of total fatty acids versus 35%. Proportions of 18:3, 20:3, and 20:4 of VPT (5.6%) and PT (6.2%) colostrum were considerably higher than T colostrum (1.8%). The fatty acid composition of cholesteryl esters of VPT, PT, and T milk was relatively similar at all subsequent lactation periods. Fatty acids esterified with cholesterol in weight percents were as follows: 10:0, 0.7; 12:0, 2.6; 14:0, 2.3; 16:0, 11.4; 16:1, 5.0; 18:0, 8.8; 18:1, 32.9; 18:2, 30.6; 18:3, 1.7; 20:3, 0.9; and 20:4, 1.8. Unsaturated fatty acids contributed 73 wt % of fatty acids in cholesteryl esters, which is considerably higher than in milk triglycerides. The greatest difference occurred in 18:2 content, which was 30.6% in cholesteryl esters and only 13.0% of total fatty acids in milk. Results suggest that unsaturated fatty acids are associated preferentially with the cholesteryl ester fraction and that the fatty acid composition of cholesteryl esters differs from the composition of total milk lipid.