AbstractMonoacylglycerol lipase activity was characterized in a soluble preparation from rabbit aorta (intima‐media) obtained by combining a 100,000×g supernatant fraction with activity solubilized from the 100,000×g precipitate fraction by treatment with Triton X‐100. Rates of hydrolysis with 1‐monoolein and 2‐monoolein substrates were nearly identical. 1‐Monoolein was a competitive inhibitor (Ki 65 μM) of 2‐monoolein hydrolysis. 2‐Monoolein and 2‐monopalmitin were both hydrolyzed more rapidly than 2‐monoarachidonin. Lipase activity measured with a 2‐monoolein substrate was inhibited by the addition of oleate, NaF and CaCl2 to the assay. Preincubation of the lipase preparation with p‐bromophenacyl bromide resulted in a potent inhibition of lipase activity; this inhibition could be prevented by dithiothreitol.