Heme-binding protein 23 (HBP23) is a cytosolic protein that binds the prooxidant heme with high affinity and has been implicated in the cellular protection against reactive oxygen species (ROS). Because lipopolysaccharide (LPS) stimulates macrophages to produce large amounts of ROS the gene expression of HBP23 was analyzed during treatment with LPS in cultured rat Kupffer cells (KC). HBP23 was constitutively expressed in KC and up-regulated on the protein and messenger RNA (mRNA) level by LPS with a time response distinct from that of TNFα, but in coordination with that of heme oxygenase-1 (HO-1), which is the inducible isoform of the rate-limiting enzyme of heme degradation. A parallel up-regulation of HBP23 and HO-1 mRNA by LPS was also observed in cultured peritoneal macrophages and peripheral blood monocytes. HBP23 mRNA induction by LPS occurred on the transcriptional level as indicated by blocking with actinomycin D. The induction of HBP23 mRNA expression by LPS was preceded by that of the inducible nitric oxide synthase (iNOS) and the production of nitrite in KC. Treatment with the NOS inhibitor NG-monomethyl l -arginine prevented HBP23 mRNA induction by LPS, which was reversed by an excess of l -arginine. Both the nitric oxide (NO)-donor S-nitroso-N-acetylpenicillamine and the peroxynitrite donor SIN-1 increased HBP23 mRNA expression. HBP23 mRNA induction by LPS was down-regulated by interleukin 10 and transforming growth factor β1 with a NO-independent mechanism. LPS-stimulated KC exhibited marked protection against the cytotoxicity mediated by H2 O2 . The data suggest that NO and peroxynitrite are major mediators of the LPS–dependent up-regulation of HBP23 in KC.