A method of regiospecific analysis of triacylglycerols (TAGs) in vegetable oils and animal fats is reported here using the electrospray ionization-mass spectrometry (MS(3)) of TAG-lithiated adducts. The fragment ions of the MS(3) from the loss of fatty acids at the sn-2 position as alpha,beta-unsaturated fatty acids were used for regiospecific identification and quantification. The ratio of the regiospecific TAGs, ABA and AAB, in an oil sample usually fraction collected by high-performance liquid chromatography can be determined by the abundance of the fragment ions of [ABA + Li-ACOOH-B'CH=CHCOOH]+ and [AAB + Li-ACOOH-A'CH=CHCOOH]+. The method was used to analyze regiospecific TAGs in extra virgin olive oil. The results showed that the saturated fatty acids, palmitic and stearic acids, were mostly located at the sn-1,3 positions and unsaturated fatty acids, oleic and linoleic acids, were mostly located at the sn-2 position.