To investigate the effects of an omega-3 fatty acid-enriched ration on the in vitro response of equine monocytes to endotoxin, an 8-week feeding trial was conducted in which linseed oil served as the source of the omega-3 fatty acid, alpha-linolenic acid. One group of horses was fed a control pelleted ration and the other group was fed an 8% linseed oil-enriched pelleted ration. After 8 weeks of feeding, monocytes were isolated and incubated in the presence of Escherichia coli O55:B5 endotoxin for 6 hr. After 8 weeks on the rations, the mean procoagulant activity and thromboxane B2 production by endotoxin-stimulated monocytes from horses consuming the linseed oil ration decreased by 51% and 71%, respectively, compared with cells from horses consuming the control ration. There was no difference in monocyte synthesis of 12-hydroxyeicosatetraenoic acid or leukotriene B4 between groups. Fatty acid analysis of membrane phospholipids revealed a decrease in the omega-6:omega-3 ratio in monocytes from horses consuming the linseed oil ration. These data suggest that dietary supplementation with alpha-linolenic acid may modify the response to endotoxin by reducing the synthesis of potentially harmful cellular mediators.