N-Acylethanolamine phospholipids (NAPEs) serve as endogenous precursors of N-acylethanolamines (NAEs), e.g. N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine that are endogenous ligands of cannabinoid receptors. Under physiological conditions, NAPE is found in very low concentrations in mammalian tissue (3-12 nmol g ). However, pathophysiological conditions may increase the endogenous NAPE levels, which again may cause an increase in endocannabinoid concentrations. This paper presents a simple and selective method for the determination of NAPE standards using negative electrospray ionization mass spectrometry (ESI-MS). The procedure provides complete positioning of all acyl and alkenyl groups contained in each NAPE species. The calibration curve for standard NAPE was linear over the range 100 fmol-50 pmol (0.1-50 ng) per injection. The lower limit of detection (signal-to-noise ratio of 3) was 100 fmol, implying that this method is superior to previous methods for the determination of NAPE. These results suggest that this ESI-MS method can be used to identify and quantify NAPE species in mammalian tissues and provide information on the corresponding NAEs to be released from the endogenous NAPE pool.