Exogenous nitric oxide (NO) suppresses endothelium-derived NO production. We were interested if this is also the case in flow-induced endothelium-derived NO production. And if so, is the mechanism due to intracellular depletion of tetrahydrobiopterin (BH₄; a cofactor of NO synthase: NOS), which results in superoxide production by uncoupled NOS? Isolated canine femoral arteries were perfused with 100 µM S-nitroso-N-acetylpenicillamine (SNAP; an NO donor) and/or 64 µM BH₄. Perfusion of SNAP suppressed flow-induced NO production, evaluated as a change in the slope of the linear relationship between perfusion rate and NO production rate (P < 0.02 vs. control; n=7). Subsequent BH₄ perfusion returned the slope to the control level. Concomitant perfusion of SNAP and BH₄ retained the control-level NO production (n = 7). Concomitant perfusion of SNAP and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; 1 mM; a membrane-permeable superoxide scavenger) also retained the control-level NO production (n = 7), while perfusion of Tiron after SNAP could not return the NO production to the control level (P < 0.02 vs. control; n = 7). We also found a significant decrease in BH₄ concentration in the endothelial cells after SNAP perfusion. In conclusion, these results indicate that exogenous NO suppresses the flow-induced endothelium-derived NO production by superoxide released from uncoupled NOS due to intracellular BH₄ depletion.