The triglycerides of the fat globules of human milk were resolved by thin-layer chromatography on silica gel impregnated with silver nitrate. The chemical structure of the glycerides was determined by gas chromatography of the glycerides and fatty acids and by a stereospecific analysis of total milk fat sample. The major triglycerides of human milk fat contained 48–54 acyl carbons which were made up of C12–C22 acids of various degrees of unsaturation. The fully saturated triglycerides (6–8%) contained mainly sn-glycerol 1-stearate 2-palmitate esterified at position 3 with C14–C18 acids. The monoenoic triglycerides (25–27%) were comprised mainly of sn-glycerol 1-oleate 2-palmitate esterified at position 3 to C12–C18 acids, as well as their racemates. The dienes (24–33%) were made up largely of sn-glycerol 1-stearate 2,3-dioleate and 2-palmitate 1,3-dioleate. The trienes (18–20%) contained mostly sn-glycerol 1,2,3-trioleate and 1-oleate 2-palmitate 3-linoleate. The tetraenes (7–21%) were identified as mainly sn-glycerol 1,2-dioleate 3-linoleate and 1-palmitate 2,3-dilinoleate. Arachidonic acid was found in the polyene fraction (6%) in the 2-position, while the linolenic acid was preferentially associated with position 3 of the tetraenes.