The stress-inducible gene heme oxygenase-1 (HO-1) provides protection against oxidative stress. Although the mechanisms by which HO-1 exerts its cytoprotection are not clearly understood, it has been speculated that carbon monoxide (CO), a catalytic byproduct following heme catabolism by HO-1, may mediate cellular cytoprotection via its anti-inflammatory properties. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a potent cytokine generated in response to bacterial endotoxin (lipopolysaccharide [LPS]) to stimulate proliferation, maturation, and effector functions of leukocytes, contributing to the proinflammatory responses to LPS. We hypothesized that HO-1 and/or CO could regulate the expression and production of GM-CSF. HO-1 overexpression, as well as exposure to a low concentration of CO, inhibited LPS-induced GM-CSF production in macrophages. Furthermore, CO inhibited LPS-induced GM-CSF induction via inhibition in the activation of the transcription factor NF-kappaB. CO inhibited LPS-induced activation of NF-kappaB, which has been shown to regulate GM-CSF transcription, by preventing the phosphorylation and degradation of the regulatory subunit IkappaB-alpha. These data raise the intriguing possibility that CO at low concentrations may play an important role in inflammatory disease states and thus has potential therapeutic implications.