A modified method for the stereospecific analysis of triacylglycerols (TGs) is presented. Partially hydrolyzed products of TG with ethyl magnesium bromide were converted to 3, 5-dinitrophenylurethane (3, 5-DNPU) derivatives, and recemic 1-and 2-monoacylglycerol (MG) derivatives were separated by thin layer chromatography (TLC) on silicic acid plates. The 1-MG fraction was separated into sn-1 and sn-3 enantiomers by high performance liquid chromatography (HPLC) on a chiral stationary phase; N- (R) -1- (α-naphthyl) ethylaminocarbonyl- (S) -valine which was chemically bound to γ-aminopropyl silanized silica. Total TG, sn-1, sn-2 and sn-3 MG fractions were converted to the methyl esters, and each of which was analyzed by gas chromatography (GC) with an opentubular column. Acyl distribution at sn-1, sn-2 and sn-3 positions was determined based on the results of GC analysis. A cacao fat sample was analyzed by this method. Direct derivation prior to fractionation was found effective for reducing isomerization of MGs.