There is evidence that NO can regulate CO production, however less is known about CO regulation of NO synthesis. Our studies were undertaken to define how CO regulates iNOS in cultured hepatocytes. CO (250ppm) exposure resulted in a significant decrease in iNOS protein, nitrite production, level of active iNOS dimer and cytosolic iNOS activity in cells stimulated with cytokines (IL-1beta) or transfected with the human iNOS gene. However, IL-1beta-stimulated iNOS mRNA expression was unaffected by CO. These effects of CO on iNOS protein levels were inhibited when CO was scavenged using hemoglobin. HO-1 induction with an adenoviral vector carrying HO-1 showed a decrease in total iNOS protein, nitrite production, and iNOS dimer level from cells stimulated by IL-1beta. iNOS protein level was significantly higher in lung endothelial cells isolated from HO-1 knockout mice compared to wild type cultures stimulated with cytokines mixture. CO was found to increase p38 phosphorylation and p38 inhibition using SB203580 increased iNOS protein levels in response to IL-1beta. Interestingly, proteasome inhibitors (MG132 and Lactacystin) and an autophagy inhibitor (3-methyladenine) reversed CO influence iNOS levels. Our results imply that CO exposure decreases NO production by suppressing dimer formation and increasing iNOS degradation through a process involving p38 activation.