The effects of anandamide, 2-arachidonoylglycerol and related compounds on the specific binding of a radiolabeled cannabinoid receptor [³H]CP55940, to synaptosomal membranes were examined. Anandamide, an endogenous cannabinoid receptor ligand, reduced the specific binding of [³H]CP55940 to synaptosomal membranes in a dose-dependent manner: the Ki value was 89 nM. 2-Arachidonoylglycerol was also shown to bind appreciably to the cannabinoid receptor in competitive inhibition experiments. The apparent binding affinity was markedly increased when the binding assay was carried out in the presence of the esterase inhibitor DFP or at 0°C. Free arachidonic acid and N-palmitoylethanolamine were almost inactive in terms of binding to the cannabinoid receptor in synaptosomal membranes. 2-Arachidonoylglycerol may be an endogenous cannabinoid receptor ligand in the brain.