Nitric oxide (NO) activates cyclooxygenase (COX) enzymes. This study shows that NO, produced by nitric oxide synthase (NOS), enhances COX activity, particularly COX-2, in mouse macrophage cells (RAW264.7) and human fetal fibroblasts. Induction of NOS and COX-2 by lipopolysaccharide (LPS) increases the release of nitrite (NO₂⁻) and prostaglandin E₂ (PGE₂). Inhibitors of NOS, such as L-NMMA or aminoguanidine, block this increase, while exogenous NO, delivered via sodium nitroprusside or glyceryl trinitrate, enhances COX activity. These effects are reversed by hemoglobin, which binds and inactivates NO, but not by methylene blue, an inhibitor of soluble guanylate cyclase. NO also increases PGE₂ production by COX-1 and COX-2 in vitro. These findings suggest that NO directly interacts with COX to enhance its activity, independent of cGMP. The study demonstrates that NO can activate COX in the absence of endogenous L-Arg, indicating a direct interaction between NO and COX. This interaction may contribute to the production of proinflammatory prostaglandins, potentially exacerbating inflammatory responses. The results highlight the role of NO in modulating COX activity, which could have implications for inflammatory diseases.Nitric oxide (NO) activates cyclooxygenase (COX) enzymes. This study shows that NO, produced by nitric oxide synthase (NOS), enhances COX activity, particularly COX-2, in mouse macrophage cells (RAW264.7) and human fetal fibroblasts. Induction of NOS and COX-2 by lipopolysaccharide (LPS) increases the release of nitrite (NO₂⁻) and prostaglandin E₂ (PGE₂). Inhibitors of NOS, such as L-NMMA or aminoguanidine, block this increase, while exogenous NO, delivered via sodium nitroprusside or glyceryl trinitrate, enhances COX activity. These effects are reversed by hemoglobin, which binds and inactivates NO, but not by methylene blue, an inhibitor of soluble guanylate cyclase. NO also increases PGE₂ production by COX-1 and COX-2 in vitro. These findings suggest that NO directly interacts with COX to enhance its activity, independent of cGMP. The study demonstrates that NO can activate COX in the absence of endogenous L-Arg, indicating a direct interaction between NO and COX. This interaction may contribute to the production of proinflammatory prostaglandins, potentially exacerbating inflammatory responses. The results highlight the role of NO in modulating COX activity, which could have implications for inflammatory diseases.