YC-1 is a newly developed agent that inhibits platelet aggregation and vascular contraction. Although its effects are independent of nitric oxide (NO), it mimics some of the biological actions of NO. For example, it stimulates soluble guanylate cyclase (sGC) and increases intracellular cGMP concentration. Here, we tested the possibility that YC-1 inhibits hypoxia-inducible factor (HIF)-1-mediated hypoxic responses, as does NO. Hep3B cells were used during the course of this work to observe hypoxic induction of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the effects of YC-1 were compared with those of a NO donor, sodium nitropurruside (SNP). In hypoxic cells, YC-1 blocked the induction of EPO and VEGF mRNAs, and inhibited the DNA-binding activity of HIF-1. It suppressed the hypoxic accumulation of HIF-1alpha, but not its mRNA level. It also reduced HIF-1alpha accumulation induced by cobalt and desferrioxamine. Treatment with antioxidants did not recover the HIF-1alpha suppressed by YC-1. We examined whether these effects of YC-1 are related to the sGC/cGMP signal transduction system. Two sGC inhibitors examined failed to block the effects of YC-1, and 8-bromo-cGMP did not mimic actions of YC-1. The effects of YC-1 on the hypoxic responses were comparable with those of SNP. These results suggest that YC-1 and SNP suppressed the hypoxic responses by post-translationally inhibiting HIF-1alpha accumulation. The YC-1 effect may be linked with the metal-related oxygen sensing pathway, and is not due to the stimulation of sGC. This observation implies that the inhibitory effects of YC-1 on hypoxic responses can be developed to suppress EPO-overproduction by tumor cells and tumor angiogenesis.