Carbon monoxide-releasing molecules are emerging as a new class of pharmacological agents that regulate important cellular function by liberating CO in biological systems. Here, we examined the role of carbon monoxide-releasing molecule 3 (CORM-3) in modulating neuroinflammatory responses in BV-2 microglial cells, considering its practical application as a novel therapeutic alternative in the treatment of stroke. BV-2 microglia cells were incubated for 24 h in normoxic conditions with thrombin alone or in combination with interferon-gamma to simulate the inflammatory response. Cells were also subjected to 12 h of hypoxia and reoxygenated for 24 h in the presence of thrombin and interferon-gamma. In both set of experiments, the anti-inflammatory action of CORM-3 was evaluated by assessing its effect on nitric oxide production (nitrite levels) and tumor necrosis factor (TNF)-alpha release. CORM-3 (75 microM) did not show any cytotoxicity and markedly attenuated the inflammatory response to thrombin and interferon-gamma in normoxia and to a lesser extent in hypoxia as evidenced by a reduction in nitrite levels and TNF-alpha production. Inactive CORM-3, which does not liberate CO and is used as a negative control, failed to prevent the increase in inflammatory mediators. Blockade of endogenous CO production by tin protoporphyrin-IX did not change the anti-inflammatory activity of CORM-3, suggesting that CO liberated from the compound is responsible for the observed effects. In addition, inhibition of the mitogen-activated protein kinases phosphatidyl inositol 3 kinase and extracellular signal-regulated kinase amplified the anti-inflammatory effect of CORM-3. These results suggest that the anti-inflammatory activity of CORM-3 could be exploited to mitigate microglia activity in stroke and other neuroinflammatory diseases.