N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 ؎ 89 ng g ؊1 fresh weight in pea (Pisum sativum cv early Alaska) to 1,608 ؎ 309 ng g ؊1 fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, with N-linoleoylethanolamine (NAE18:2) being the most abundant (approaching 1,000 ng g ؊1 fresh weight in cottonseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35°C in 50 mM MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5,5-dithio-bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K m of 65 M and a V max of 83 nmol min ؊1 mg ؊1 protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the family of "endocannabinoids," which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.