The sn-1,2-, sn-2,3-, and X-1,3-diacylglycerols derived by Grignard degradation of purified menhaden oil triacylglycerols were isolated by conventional thin-layer chromatography with boric acid complexing. The sn-1,2(2,3)-diacylglycerols were resolved into sn-1,2- and sn-2,3-diacylglycerols by stepwise digestion with phospholipase C of the corresponding phosphatidylcholines and the positional distribution of the fatty acids were determined. Diacylglycerols were converted into trimethylsilyl ethers and resolved on the basis of molecular weight and degree of unsaturation by gas–liquid chromatography using a polar capillary column and isothermal or programmed temperatures. The order of chromatographic elution was established for more than 70 major and minor species by reference to primary and secondary diacylglycerol standards and by calculation of relative retention times. The identified molecular species ranged in carbon number from 28 to 44 and in double bond number from 0 to 12 being made up of C14–C22 fatty acids with 0 to 6 double bonds each and representing the n – 9, n – 6, n – 4, n – 3, and n – 1 series. The gas–liquid chromatographic determinations yielded proportions of all major species that were consistent with those calculated from the knowledge of the stereospecific distribution of the fatty acids in the original triacylglycerol molecules.Key words: Grignard degradation, rac-phosphatidylcholines, phospholipase C, enantiomeric diacylglycerols, thin-layer chromatography, molecular species of diacylglycerols, composition of fatty acids.