Leafy spurge (Euphorbia esula L.), a plant introduced into the Great Plains of North America from Europe, has become a serious economic and ecological threat to the productivity of agricultural and natural areas. Cattle, the predominant livestock species on the Great Plains, as well as common wild ruminant species in this region appear to consume little if any leafy spurge. This is likely because they experience a toxic response after consuming small amounts of this plant, and they consequently learn to avoid it. Domestic sheep and goats can consume considerable amounts of leafy spurge and are used to help control it, but even these species may suffer a toxic response at high levels of intake. Toxic diterpene ingenols have been isolated from leafyspurge tissues, but compounds in this plant have not been evaluated with respect to their capacity to induce food aversion learning in ruminants. We conducted bioassay-guided fractionations of leafy spurge in an attempt to isolate toxic and aversive compounds. These bioassay-guided fractionations led to identification of ingenol and one of its diesters as two toxic compounds in leafy spurge that are potentially aversive to cattle.