The distribution of fatty acids esterified at each position of glycerol in phosphatidyl serine (PS), phosphatidyl inositol (PI), and diphosphatidyl glycerol (DPG) and the composition of sphingomyelin (SPH), lysophosphatidyl choline, and free ceramides isolated from rat liver was determined. The carbon number distribution of the diglyceride acetates derived from each of the phosphoglycerides and of the ceramide trimethylsilyl ether derivatives was also determined. The rat liver SPH contained a high percentage of long-chain saturated and monounsaturated acids. The carbon number distribution of free ceramides and those derived from SPH was similar. Each of the phosphatides exhibited a characteristic acyl distribution and diglyceride carbon-number distribution. The 1-position of PS and PI contained almost exclusively (> 90%) stearic acid. Unlike the other phosphatides, DPG contained primarily C-18 acids and a high percentage of the major acid, 18:2, was esterified at the 1-position. The diglycerides derived from PI and DPG had or approached a 1-random-2-random distribution, whereas those of PS showed pairing of certain acids. The data indicate that selectivity of diglyceride species characterizing each phosphoglyceride class occurs in rat liver. None of the carbon number distributions of the diglycerides derived from each of the phosphoglycerides resembled the 1,2-diglycerides obtained from rat liver triglycerides. © 1969.