Anandamide, an endogenous canabinoid substance, is hydrolyzed by an amidohydrolase activity present in rat brain and liver. We report that the bromoenol lactone, (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BTNP), is a potent inhibitor of this enzyme activity. BTNP prevented anandamide hydrolysis in rat brain microsomes with an IC50 of 0.8 +/- 0.3 microM. Kinetic and dialysis experiments indicated that this effect was non-competitive and irreversible. After chromatographic fractionation of the enzyme activity, BTNP was still effective, suggesting that it interacts directly with the enzyme. Anandamide hydrolysis was 12-fold greater in rat cortical neurons (1.94 +/- 0.1 pmol/min/mg protein) than in cortical astrocytes (0.16 +/- 0.01 pmol/min/mg protein) and, in either cell type, it was inhibited by BTNP (IC50 = 0.1 microM in neurons). These results suggest that BTNP may provide a useful lead for the development of novel inhibitors of anandamide hydrolysis.