Widespread expression of the alpha-subunit of hypoxia-inducible factor (HIF-1alpha) was observed in samples of human oral squamous cell carcinoma. In all the cases, this was accompanied by a widespread distribution of nitric oxide (NO) synthases (NOS). Furthermore, in three human cell lines derived from human oral squamous cell carcinoma, the accumulation of HIF-1alpha was prevented either by inhibition of NOS activity with the nonspecific NOS inhibitor N(G)-monomethyl-L-arginine or by the antioxidants N-acetyl-L-cysteine and ascorbic acid. We suggest that, in certain forms of cancer, NO might be responsible for the accumulation of HIF-1alpha by a mechanism dependent on free radicals.