N-acyl phosphatidylethanolamine (N-acyl PE) and free N-acylethanolamine (NAE) in mouse peritoneal macrophages were identified and quantified by gas chromatography-mass spectrometry (GC-MS) of tertbutyldimethylsilyl derivatives in the presence of internal standards synthesized from [1,1,2,2-2H4]ethanolamine. N-acyl PE was present at a level of 123-187 pmol/mumol lipid P (521-768 pmol/10(8) cells), with arachidonic acid making up about 3-4% of the N-acyl moieties. NAE, on the other hand, was present at a level of only 17-30 pmol/mumol lipid P (70-121 pmol/10(8) cells), with N-arachidonoylethanolamine (anandamide) making up less than 1% of total NAE. Use of deuterium labeled internal standards and optimization of GC-MS conditions makes it possible to detect as little as 0.1 ng of saturated and 1 ng (3 pmol) of polyunsaturated NAEs in a lipid extract. The present method can be used to determine agonist-induced changes in the levels and compositions of N-acyl PE and NAE.