Inducible nitric oxide (NO) synthase (NOS) II has been implicated in macrophage-mediated antitumor activity. However, use of the NOS II gene in cancer therapy is problematic because of the double-edged nature of NO action. Herein we show that adenoviral vectors mediated effective NOS II gene transfer into various human tumors. Production of NO significantly up-regulated multiple angiogenic molecules. However, the NO-producing tumor cells did not form tumors or metastases in ectopic or orthotopic xenograft nude mouse models. The dramatic loss of malignancy was due to NO-mediated apoptosis. We also generated a series of adenoviral vectors harboring mutant NOS II genes that expressed mutant NOS II proteins with defined levels of enzymatic activity. Tumor cells transduced with these NOS II genes produced NO at different levels, which directly correlated with the antitumor activity in vitro and in vivo. This demonstration using a relevant biological system shows that NO produces dose-dependent antitumor activity in vitro and in vivo, regardless of its up-regulation of protumor factors.