Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH₄), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH₄ levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH₄ biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear. We sought to investigate the signaling pathways whereby cytokines induce GTPCH I expression in human umbilical vein endothelial cells (HUVECs). Interferon-γ (IFN-γ) induced endothelial cell GTPCH I protein and BH₄ modestly, whereas high-level induction required combinations of IFN-γ and tumor necrosis factor-α (TNF-α). In the presence of IFN-γ, TNF-α increased GTPCH I mRNA in a manner dependent on nuclear factor-κB (NF-κB), as this effect was abrogated by overexpression of a dominant-negative IκB construct. HUVEC IFN-γ treatment resulted in signal transducer and activator of transcription 1 (Stat1) activation and DNA binding in a Jak2-dependent manner, as this was inhibited by AG490. Conversely, overexpression of Jak2 effectively substituted for IFN-γ in supporting TNF-α–mediated GTPCH I induction. The role of IFN-γ was also Stat1-dependent as Stat1-null cells exhibited no GTPCH I induction in response to cytokines. However, Stat1 activation with oncostatin M failed to support TNF-α–mediated GTPCH I induction because of concomitant Stat3 activation. Consistent with this notion, siRNA-mediated Stat3 gene silencing allowed oncostatin M to substitute for IFN-γ in this system. These data implicate both NF-κB and Stat1 in endothelial cell cytokine-stimulated GTPCH I induction and highlight the role of Stat3 in modulating Stat1-supported gene transcription. Thus, IFN-γ and TNF-α exert distinct but cooperative roles for BH₄ biosynthesis in endothelium that may have important implications for vascular function during vascular inflammation.