The 5-lipoxygenase (5-LO) catalyzed formation of leukotriene (LT) lipid mediators is a pathway contributing to inflammatory events in asthma and more recently has been associated with cardiovascular disease. However, the relative impact of this pathway in atherogenesis has been controversial and a variety of mixed results reported. The goal of these studies was to assess the importance of the 5-LO/LT pathway in mice with either genetic (5-LO(-/-)) or pharmacological (L-739,010) inhibition of the 5-LO pathway on an apolipoprotein E deficient (apoE(-/-)) background when subjected to either an 8-week (Paigen) or 6 months (Western) atherosclerotic diet regimen. Atherosclerotic lesion analysis at the aortic root, brachiocephalic artery and throughout the whole aorta by en face Sudan IV staining was determined, as well as blood lipid levels. Ex vivo calcium ionophore-stimulation of whole blood demonstrated a significant reduction in the capacity to form LTB(4) in 5-LO(-/-) and drug-treated 5-LO(+/+) mice. Quantitative analysis of atherosclerotic lesions did not differ between groups at all three sites. Moreover, the composition of advanced lesions in the brachiocephalic arteries did not indicate altered plaque disruption as a result of 5-LO gene inactivation. These results do not support a role for the 5-LO/LT pathway in intermediate to advanced atherosclerotic lesion development in mice.