Increased monocyte adhesion to aortic endothelium is observed in the pathogenesis of atherosclerosis. The role of endothelial acyl-coenzyme A:cholesterol-acyltransferase (ACAT) in the regulation of monocyte adhesion is not known. To examine the potential role of this enzyme in monocyte adhesion, a specific ACAT inhibitor, CI-976, was utilized. Although the basal adhesion of U937 monocytic cells to porcine aortic endothelial cells was low, treatment of the endothelial cells with lipopolysaccharide (LPS) markedly increased monocyte adhesion. Monocyte adhesion to LPS-treated endothelial cells was markedly inhibited by CI-976 treatment of the endothelial cells. Similarly, another ACAT inhibitor, PD 132301-2, whose structure is distinct from CI-976, also decreased monocyte adhesion. CI-976 treatment of endothelial cells also decreased endothelial cell ACAT activity. Since leukotriene B4 (LTB4) is known to promote leukocyte-endothelial cell adhesion, endothelial cell production of this leukotriene was examined after incubation with CI-976. CI-976 treatment markedly decreased LTB4 synthesis. Exogenous LTB4 addition to CI-976 treated cells reversed the effects of this compound on monocyte adhesion. These data demonstrate that ACAT inhibitors decrease monocyte adhesion to endothelial cells. Similar mechanisms may contribute to antiatherosclerotic effects of ACAT inhibitors in vivo.