A fragment of DNA sequence derived from a hepatotropic virus, named NV-F was isolated recently. The aim of this study was to examine whether this virus was associated with hepatocellular carcinoma (HCC). Total cellular DNA was extracted from hepatocellular carcinoma tissues. NV-F virus DNA was detected by PCR. The PCR products were subjected to sequence analysis. Of the 78 HCC samples included, 12 (15.4%) were positive for NV-F virus DNA. Sequence analysis of the 12 amplified DNA fragments revealed a point mutation in one of them. The clinicopathological parameters between patients with and without NV-F virus infection were compared. It was found that patients with NV-F virus infection were older than those without NV-F virus infection (mean ages, 61.5 versus 52.5 years; P = 0.032). Otherwise, no difference was observed between the two groups. Of the 12 HCC patients positive for NV-F virus DNA, 11 patients were co-infected by either hepatitis B or C virus. The remaining patient was a Taiwanese aboriginal inhabitant with cryptogenic cirrhosis. In conclusion, NV-F virus DNA was identified in 15.4% of HCC tissues. HCC patients with NV-F virus infection were significantly older than those without NV-F virus infection.