Flavonoids affect the inflammatory process of the mammalian system and possess anti-inflammatory as well as immunomodulatory activities in vitro and in vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of various naturally occurring flavonoids on NO production in LPS-activated RAW 264.7 cells were evaluated in vitro. Flavonoids such as apigenin, wogonin, luteolin, tectorigenin, and quercetin inhibited NO production, as measured by nitrite formation at 10–100 μM. The most active among 26 flavonoid derivatives tested were apigenin, wogonin, and luteolin, having ₅₀ values of 23, 17, and 27 μM, respectively, while AMT, a synthetic selective iNOS inhibitor, had an₅₀ value of 0.09 μM. In contrast, flavanones, such as naringenin, and flavonoid glycosides, such as apiin, did not demonstrate significant inhibition up to 100 μM. These results clearly indicated that a C-2,3 double bond might be important, and that the potency of inhibition depended upon the substitution patterns of the flavonoid molecules. The inhibitory activity of flavonoids was not due to direct inhibition of iNOS enzyme activity because they did not reasonably inhibit iNOS activity, as measured by [³H]citrulline formation from [³H]arginine, up to 100 μM. In contrast, wogonin and luteolin concentration-dependently reduced iNOS enzyme expression, when measured by western blotting, at 10–100 μM. All these results clearly demonstrated that certain flavonoids inhibit NO production in lipopolysaccharide-activated RAW 264.7 cells, and their inhibitory activity might be due to reduction of iNOS enzyme expression.