The formation of anandamide (N-arachidonoylethanolamine), N-acylethanolamine, and N-acylphosphatidylethanolamine was studied in primary cultures of rat cortical neurons. The cells were incubated for 22 h with [C]ethanolamine, [U-C]arachidonic acid, [H]arachidonic acid, [P]phosphate, [C]stearic acid, or [H]myristic acid. The lipids from the cells and media were separated by thin layer chromatography. [C]Ethanolamine labelling revealed two compounds (I and II), which on different thin layer chromatography systems migrated as N-acylethanolamine (0.06-0.55% of total radioactivity) and N-acylphosphatidylethanolamine (0.66-6.49% of total radioactivity), respectively. Compound II was also labelled with [P]phosphate, and radioactive fatty acids. Treatment of compound II with phospholipase D (Streptomyces chromofuscus) resulted in two compounds, one comigrating as phosphatidic acid and the other as N-acylethanolamine. Compound I could be labelled with [C]stearic acid and [H]myristic acid, but not with [H]- or [C]arachidonic acid. Exogenous [H]anandamide was metabolised with a t( 1/2 ) of 2.6 h. The labelling of the two compounds identified as N-acylethanolamine and N-acylphosphatidylethanolamine were more pronounced the older the culture. The neurotoxic amino acid, glutamate, stimulated within 2 h dose-dependently (ED = 40 µM) the formation of both compounds. It is suggested that N-acylethanolamine and N-acylphosphatidylethanolamine are formed in relation to the cytotoxicity induced by glutamate, and that these compounds may be markers of neurotoxicity. We could not detect any formation of anandamide using radioactive arachidonic acid.