We studied the effects of cAMP on cytokine (interferon-γ plus tumor necrosis factor-α)-induced stimulation of tetrahydrobiopterin (BH₄) synthesis in human umbilical vein endothelial cells (HUVEC). The cytokine mixture caused a marked increase in the biosynthesis and release of BH₄ by HUVEC. Dibutyryl-cAMP produced a dose-dependent inhibition of this cytokine-induced stimulation of synthesis and release of BH₄ by these cells. 8-Bromo-cAMP also caused a significant inhibition, although the effects were less marked than those of dibutyryl-cAMP. Both forskolin and the stable analog of prostacyclin, iloprost, caused cAMP accumulation and a concomitant diminution of the cytokine-induced BH₄ synthesis in HUVEC. Dibutyryl-cAMP and iloprost also significantly inhibited the cytokine-induced stimulation of GTP cyclohydrolase I (GCHI) activity and mRNA production. We concluded that the suppression by the cAMP messenger system of cytokine-induced stimulation of synthesis and release of BH₄ by HUVEC can be attributed to the inhibition of the activity of GCHI, the rate-limiting enzyme in BH₄ biosynthetic pathway, in HUVEC. The data also suggest that the cAMP-mediated reduction in the GCHI mRNA level may at least partially explain the decline in GCHI activity. It is reasoned that under inflammatory conditions, cAMP-elevating agents such as prostacyclin exert regulatory effects on circulation by inhibiting cytokine-induced synthesis and release of BH₄ by HUVEC.