Stearoylethanolamide (SEA) is present in human, rat and mouse brain in amounts comparable with those of the endocannabinoid anandamide (arachidonoylethanolamide; AEA). Yet, the biological activity of SEA has never been investigated. We synthesized unlabelled and radiolabelled SEA to investigate its binding, degradation and biological activity in rat C6 glioma cells. We report that SEA binds to a specific site distinct from known cannabinoid or vanilloid receptors, and that AEA and capsazepine partly (approx. 50%) antagonized this binding. Treatment of C6 cells with SEA inhibits cellular nitric oxide synthase and does not affect adenylate cyclase, whereas treatment with cannabinoid type 1 agonist 2-arachidonoylglycerol activates the former enzyme and inhibits the latter. C6 cells also have a specific SEA membrane transporter, which is inhibited by NO, and a fatty acid amide hydrolase capable of cleaving SEA. In these cells, SEA shows pro-apoptotic activity, due to elevation of intracellular calcium, activation of the arachidonate cascade and mitochondrial uncoupling. NO further enhances SEA-induced apoptosis. Moreover, the cannabinoid type 1 receptor-mediated decrease in cAMP induced by AEA in C6 cells is potentiated by SEA, suggesting that this compound also has an 'entourage' effect. Taken together, this study shows that SEA is an endocannabinoid-like compound which binds to and is transported by new components of the endocannabinoid system. It seems noteworthy that degradation and pro-apoptotic activity of SEA are regulated by NO in a way opposite to that reported for AEA.