The purpose of this study was to characterize the lipolytic activities released by heparin from rat livers. Heparin perfusates of rat livers degraded mono-oleoylglycerol, trioleoylglycerol and phosphatidylcholine in emulsions as well as in chylomicrons, chylomicron remnants, low-density lipoprotein/high-density lipoprotein-1 (LDL/HDL-1) and high-density lipoprotein-2 (HDL-2). The preferred substrate was mono-oleoylglycerol. Heparin perfusates were separated by chromatography on either heparin-Sepharose or N-desulphated, N-acetylated heparin-Sepharose into at least two related lipases which differed in their ability to hydrolyse HDL-2 phosphatidylcholine, but not in their ability to degrade mono-oleoylglycerol, trioleoylglycerol and phosphatidylcholine in emulsions. The sodium dodecyl sulphate (SDS)/polyacrylamide-gel-electrophoretic patterns of heparin perfusates purified on either normal or N-desulphated N-acetylated heparin-Sepharose were the same, despite differences in their ability to degrade HDL-2 phosphatidylcholine. There was a single band of M(r) 56000 without 2-mercaptoethanol in the SDS disruption buffer and three major bands, of M(r) 62000, 59000 and 56000, with 2-mercaptoethanol present. When mono-oleoylglycerol lipase was purified 161-fold, there was a concomitant enrichment of the M(r)-56000 protein.