NV-F is a novel hepatotropic viruslike agent. To investigate the impact of the NV-F agent during chronic hepatitis C virus (HCV) infection, 101 consecutive patients with chronic HCV infection were evaluated. NV-F DNA in serum samples and NV-F antigen expression in liver tissues were assessed. All patients subsequently received a 6-month course of interferon-based antiviral therapy. Of the 101 patients, 30 (29.7%) were positive for serum NV-F DNA. Immunohistochemical analysis revealed positive NV-F antigen expression in the liver in 14 of these 30 patients. Patients positive for serum NV-F DNA had significantly higher serum aminotransferase levels (P < .001) and higher Knodell histology activity index values (P < .001). The sustained virological response rate for HCV clearance was not significantly different between patients with and those without detectable serum NV-F DNA. In conclusion, coinfection of the NV-F agent in chronic HCV infection is associated with more severe hepatitis activity.