Many studies have demonstrated the function of nitric oxide (NO) or nitric oxide synthase-2 (NOS-2) in cancer as pro-neoplastic or anti-neoplastic effectors, but the role of NO and NOS-2 in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the levels of NO production and NOS-2 expression in HCC and adjacent non-tumor liver tissues and to clarify whether the levels of NO/NOS-2 are related to the clinicopathological features of HCC. The levels of NO production were examined in tumor and adjacent non-tumor liver tissues of 30 patients with HCC. The expression of NOS-2 was detected by real-time polymerase chain reaction (RT-PCR) and immunohistochemical analysis in HCC and/or adjacent non-tumor liver tissues. Mutant p53 and proliferating cell nuclear antigen (PCNA) were also immunohistochemically investigated in liver tissues. The levels of NO in HCC were significantly lower compared to adjacent non-tumor liver tissues (P<0.001). The relative m RNA and protein expression levels of NOS-2 in HCC were also significantly lower compared to adjacent non-tumor liver tissues (P<0.01 for both). We found that the levels of NO in patients suffering from HCC metastasis were lower compared to those without metastasis (P<0.05) and NOS-2 expression was correlated with tumor diameter (P<0.05) and metastasis (P<0.05). In addition, mutant p53 protein was expressed in the majority of HCC samples and the proliferation rate of HCC was significantly higher than that of adjacent non-tumor liver tissues. These data indicate that decreased levels of NO/NOS-2 may partially contribute to overexpression of the mutant p53 protein and excessive proliferation; this may be a potential mechanism in the development and progression of HCC.