Long-chain N-acylethanolamines (NAEs) were assayed in pig, sheep and cow brain by gas chromatography/mass spectrometry of their tert.-butyldimethylsilyl derivatives in the presence of deuterium-labeled internal standards. Immediately after death, total NAEs ranged from about 2.7 micrograms/g wet weight (sheep, cow) to 6.5 micrograms/g wet weight (pig) and consisted almost exclusively (99%) of saturated and monounsaturated species. Anandamide (20:4n-6 NAE) comprised about 1% of total NAE in pig and cow brain, but was absent in freshly extracted sheep brain. When pig brain was analysed after 0.5, 1, 3, 4 and 23 h at ambient temperature, NAE levels were found to increase substantially over the entire time period with 20:4n-6 NAE formation exhibiting a time lag compared to that of saturated and monounsaturated NAEs.