Milk lipids were fraetionated by silicic acid column chronlatography and preparative thinlayer chromatography [TLC). Ceramide monohexoside (CMH), eeramide dihexoside (CDH), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), and ~phingomyelin (Sph) were isolated, and the purity of each was checked by infrared spectroscopy and TLC. The diacylphospholipids were hydrotyzed with phospholipase A and the products separated by TLC. Fatty acid methyl esters were prepared from the various fractions and analyzed by gas chromatography.The glycolipids, CMH and CDH, and Sph contained large amounts of long-chain saturated fatty acids, especially C2.,:., C2:~:., and C,,4:,.PE, PS, and PC contained C~o-C,,,, normal and branched-chain saturated fatty acids, and C15-'C2o unsaturated fatty acids (mainly monoenes). The distributions of saturated acids between the a'-and B-positions were respectively : PE, 46 and 11%; PS, 65 and 19%; and PC, 72 and 53%. PC was exceptional in that there was 10.8% myristie acid in the B-position and only 5.6% in the a'-position. PE and PS were similar in composition except that in PE oleic acid was evenly distributed, and in PS was largely in the B-position. In general, PC was much more saturated than PE or PS, and there was no overall pattern governing the specific distribution of the fatty acids in the three diaeylphospholipids. Comparison with PC from other bovine tissues and from egg lecithin showed that fatty acids are located much less specifically in milk phospholipids than in PC from other sources.