Highly purified lipoprotein lipase from human postheparin plasma has been incubated with [ 3H]glycerol- or [2- 14C]fatty acid-, [ 3H]glycerol-labeled trioleate and the reaction products separated with thin-layer chromatography. 1. 2. A small but constant amount of diglycerides was found among the reaction products. The 1,2-isomer constituted > 94% of the diglyceride fraction. The 1,3-isomer found was not formed by direct hydrolysis of the triglyceride 2-ester bond. 2. 3. There was an appreciable accumulation of monoglycerides. Tlie 2-isomer constituted 64–97% of this fraction. In most cases, > 90% of the 1-monoglyceride found must have been formed by isomerization from the 2-isomer. 3. 4. The principal reaction sequence was triglyceride →1,2-diglyceride →2-monoglyceride /ah 1-monoglyceride. In these reaction steps, human lipoprotein lipase therefore has positional specificity for the 1(3)-position. 4. 5. No conclusive evidence is presented for the mechanism of hydrolysis of the monoglycerides. The data support the view, however, that glycerol is obtained predominantly from 1-monoglycerides formed by isomerization.