Nitric oxide (NO) has cytotoxic effects but NO producing neurons are resistant to NO toxicity. These results suggest the presence of self-protecting factors for NO toxicity. Recently, 6R-tetrahydrobiopterin (6R-BH 4 ), a cofactor for NO synthase (NOS), has been reported to degrade NO raising the possibility that 6R-BH 4 acts as a self-protecting factor for NO toxicity. In PC12 cells which have NOS, three-day culture with sodium nitroprusside (SNP) or NOC-12, NO generators, at 10-100 M increased nitrite and nitrate concentrations in the culture medium and induced death of PC12 cells. Coadministration of 6R-BH 4 (10 or 30 M) with SNP or NOC-12 prevented cell death with reduction of nitrite and nitrate in the medium. Inhibition of 6R-BH 4 synthesis by 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor for GTP cyclohydrolase I, decreased cellular 6R-BH 4 content and viable cell number. The inhibiting effects of DAHP were restored by exogenous 6R-BH 4 . NOS activity, as estimated by nitrite concentrations in the medium, was unchanged by DAHP. Hypoxanthine and xanthine oxidase, which produce superoxide, mimicked the cell-protecting effect of 6R-BH 4 which is reported to generate superoxide during its autoxidation. These results suggest that 6R-BH 4 acts as a self-protecting factor for NO toxicity with generation of superoxide in NO-producing neurons.