We previously showed a sequence of the 5'-flanking region of the human inducible nitric oxide synthase (hiNOS) gene that included putative cis-acting elements. A reported plasmid containing the 5'-flanking region of the hiNOS gene upstream from its reporter gene was constructed, and then transiently or stably transfected into human cells known to express the hiNOS gene following cytokine stimulation. The transfected cells showed the inducibility of the reporter activity following interleukin-1beta stimulation. Reporter inducibility disappeared in cells transfected with a plasmid mutated in the putative nuclear factor (NF)-kappaB binding region. In addition the induction was inhibited by a treatment of anti-oxidant, pyrrolidinedithiocarbamate, known as an NF-kappaB inhibitor. Our results demonstrate that the promotor including the NF-kappaB region is functional and that the hiNOS gene is transcriptionally regulated via NF-kappaB activation in human cells.