Abstract The effect of the intracellular 5,6,7,8-tetrahydrobiopterin (BH 4 ) on the increased γ-glutamylcysteine synthetase (γ-GCS) mRNA expression induced by nitric oxide (NO) was investigated in RAW264.7 cells. Low doses of nitroprusside (SNP), ranging from 0.1 mM to 0.5 mM, significantly increased the intracellular glutathione levels along with the expression of mRNA for γ-GCS, a rate limiting enzyme of de novo glutathione synthesis. The increased expression was not abolished by exogenously added BH 4 itself, but by sepiapterin, a precursor of BH 4 synthesis, in a dose-dependent manner. The blockage by sepiapterin was ineffective in the presence of N-acetyl serotonin (NAS), an inhibitor of sepiapterin reductase. The increased γ-GCS mRNA expression was also inhibited by catalase, a scavenger of hydrogen peroxide. Evidence was further provided that BH 4 effectively scavenged hydrogen peroxide. These results suggest that intracellular BH 4 may play a role as an inhibitor of glutathione synthesis induced by NO via scavenging hydrogen peroxide, a mediator of the gene expression for the de novo glutathione synthesis pathway.