Heme oxygenase-1 (HO-1) catabolizes the degradation of heme into bilirubin, carbon monoxide, and iron ions. The HO-1 products provide antioxidant cytoprotection in addition to having potent antiinflammatory and immunomodulatory functions. HO-1 is induced by its substrate heme and environmental factors including oxidative and heat stresses. Although previous studies reported that lipopolysaccharide (LPS) induced the expression of both the HO-1 gene and its protein in macrophages, the major regulators of HO-1 expression remain unknown. To identify these regulators, we used two types of cell, the murine macrophage-like cell line J774.1/JA-4 and its LPS-resistant mutant, LPS1916. Based on a comparison of the results obtained with these cells, we found that nitric oxide (NO) was closely linked to the induction of HO-1. Real-time polymerase chain reaction (PCR) showed that the time course for inducible HO-1 mRNA by LPS or LPS+interferon (IFN)-γ was similar to that for inducible NO synthase (iNOS) mRNA. Furthermore, the expression of iNOS mRNA and protein increased earlier than that of HO-1 mRNA and protein. N -Nitro-L-arginine methyl ester, an NO synthase inhibitor, reduced both HO-1 expression and NO production in LPS+IFN-γ-treated JA-4 cells. Furthermore, NOC-12, an NO donor, significantly induced HO-1 expression not only in JA-4 but also in LPS1916 cells. Reactive oxygen species (ROS) scavengers, such as superoxide dismutase and catalase, did not affect HO-1 protein expression in LPS+IFN-γ-treated JA-4 cells. These results suggest that, among ROS, NO plays an important role in HO-1 induction in activated macrophages treated with LPS+IFN-γ.