The presence of monoacylglycerol lipase was established in extracts of acetone-dried powders from rat and bovine brains using thioester substrate analogs. At pH 7.4, the apparent Km and Vmax values for 1-S-decanoyl-1-mercapto-2,3-propanediol were 56 microM and 227 nmol/h/mg protein in bovine gray matter. The divalent metal ions Ca2+ and Mg2+ had no effect on enzymic activity, but Zn2+ at 500 microM produced a 50% inhibition of this enzyme. Free fatty acids also caused a marked inhibition of monoacylglycerol lipase activity. Norepinephrine and 5-hydroxytryptamine slightly stimulated the enzymic activity. Hypoxic-hypoxia and 30-s postdecapitation ischemia resulted in a considerable increase in monoacylglycerol lipase activity of rat brain. However, the increased activity of monoacylglycerol lipase returned to normal after 5 min of ischemia. The increased activity of monoacylglycerol lipase during hypoxic-hypoxia and short-time ischemia may be partially responsible for increased levels of free fatty acids during these processes.