Monoacylglycerol molecular species, as their di-3,5-dinitrophenylurethane derivatives, were well separated by normal phase high-performance liquid chromatography on nitrile-bonded phase. The peaks emerged in the order 20:0, 18:0, 16:0, 18:1, 16:1, 18:2 and 18:3. The peaks of 1- and 2-mono-acylglycerols with the same acyl group showed complete overlapping. This method could be applied to get acyl compositions in the three positions of triacyl-sn-glycerols in their stereospecific analysis.