Heme oxygenase-1 (HO-1) catalyzes the regiospecific oxidative degradation of heme to biliverdin IXα, iron, and carbon monoxide. Biliverdin IXα is subsequently reduced to bilirubin IXα by biliverdin reductase. HO-1 expression is induced under various disease conditions, including atherosclerosis, but it is unknown whether HO-1 catalyzes heme breakdown in the regions at risk. Using hypercholesterolemic rabbits fed a cholesterol-enriched diet, we attempted to demonstrate the involvement of HO-1 induction and bilirubin IXα production in atherosclerotic regions. Expression levels of HO-1 mRNA were elevated in the aortas of hypercholesterolemic rabbits. In situ hybridization and immunohistochemistry revealed that mRNA and protein of HO-1 are induced in endothelial cells and foam cells (lipid-filled macrophages) in atherosclerotic lesions. Furthermore, immunohistochemistry with the use of an anti-bilirubin-IXα monoclonal antibody, 24G7, demonstrated accumulation of bilirubin IXα in foam cells, indicating that heme is actually degraded in atherosclerotic lesions. Remarkably, bilirubin IXα, like HO-1 protein, is predominantly accumulated in the perinuclear regions of foam cells. These results provide the first in vivo evidence of the colocalization of HO-1 and bilirubin IXα in foam cells, suggesting a role of HO-1 induction in the modulation of macrophage activation in atherosclerosis.