Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH₄) is a critical determinant of eNOS activity and coupling and GTP cyclohydrolase (GCH I) is the rate-limiting enzyme in its generation. Thus, the initial purpose of this study was to determine whether decreases in BH₄ could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH I expression and BH₄ levels in the intact lamb. Contrary to our hypothesis we found that there was a significant increase in both plasma BH₄ levels and peripheral lung GCH I protein levels. Further, in vitro we found that exposure to the NO donor, spermine NONOate (SPNONO) lead to an increase in GCH I protein and BH₄ levels in both COS7- and pulmonary arterial endothelial cells. However, SPNONO-treatment also causes a significant increase in phospho-CREB levels as detected by western blot analysis and significantly increased cAMP levels as detected by enzyme immunoassay (EIA). Further, utilizing GCH I promoter fragments fused to a luciferase reporter gene we found that GCH I promoter activity was enhanced by SPNONO in a CREB dependent manner and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest NO increases BH4 levels through a cAMP-CREB mediated increase in GCH I transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH₄ levels.