In the present study, a model system for studying the role of nitric oxide (NO) in tumor growth and metastasis was reported. Incubation of Panc02 murine pancreatic adenocarcinoma cells in vitro with cytokines and interferon led to heterogeneous expression of NO synthase II (NOS II) protein. Clonal sublines expressing different levels of NOS II were then established using a limited dilution technique. After orthotopical implantation into the pancreas of syngeneic C57BL/6 mice, clones with a low level of NOS II expression produced tumors in pancreas, metastasized to the liver, and formed ascites, whereas those having a high level of NOS II expression did not. Liver-metastasis variants having low to high metastatic ability were also established using in vivo/in vitro passage. Compared with parental Panc02 cells exhibiting a high level of NOS II expression, these variants had a decreased level of NOS II expression. Furthermore, the heterogeneous Panc02 cells were injected intravenously into a large number of syngeneic mice. Variants that metastasized to the liver, lung, skin, peritoneum, ovary, and lymph nodes were established. All of the metastatic variants exhibited a lower level of NOS II expression than the parental Panc02 cell line did. However, the phenotypes of NOS II induction and metastatic ability were unstable. Multiple in vitro/in vivo selection led to stable low NOS II expression and high metastatic potential. Finally, to further confirm the role of NOS II expression derived from tumor cells in metastasis, poorly metastatic Panc02-H0 and highly metastatic Panc02-H7 cells were injected into the pancreas of syngeneic NOS II(-/-) mice, and groups of mice received i.p. injections of either phosphate-buffered saline or L-N(6)-(1-iminoethyl) lysine. Inhibition of NOS II activity in vivo significantly promoted distant liver metastasis. Collectively, these data show that NOS II expression is highly heterogeneous and dynamically regulated, which can directly influence tumor growth and metastasis.