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The role of plant lipases in the hydrolysis of dietary lipids in the rumen of pasture-fed ruminants has been investigated by means of in vitro and in vivo experiments with rumen contents. Lipases present in the leaves of numerous pasture plants remained highly active for at least 5 h in the presence of metabolising rumen microorganisms, leading to the hydrolysis of triglyceride. Parallel experiments showed that the lipase activity of actively metabolising micro-organisms in rumen fluid was very low. A slight increase in lipolytic activity attributable to micro-organisms occurred after incubations of about 4 h with plant extract when the metabolic activity of the microbial population had passed its peak. In vivo experiments showed that the lipolytic activity of rumen fluid, obtained 0.5 to 5.0 h after feeding fresh grass, was about twice that of rumen fluid obtained after overnight fasting. Paired feeding experiments showed that lipase activity was higher in rumen fluid obtained from a pasture-fed, than from a hay-fed animal. Nevertheless, there was appreciable activity in the rumen contents obtained from the hay-fed twin, which is consistent with the presence of lipase activity in the extracts of dried grass. Multiple forms of plant esterases and phosphatases were present in the soluble fraction of rumen fluid several hours after feeding. It is concluded that the rapid release of free fatty acids which follows the ingestion of pasture is due mainly to the activity of plant enzymes and that rumen microorganisms have a subsidary role in hydrolysing ingested lipid.