The antiviral activity of interferon-lambda (IFN-lambda) against several viruses has been reported, but it has not been investigated whether human IFN-lambda can affect replication of the hepatitis B virus (HBV) in human hepatocyte-derived cells. We found that replication of HBV in a human hepatoma cell line was reduced by approximately 30% following treatment with a high concentration of IFN-lambda. Furthermore, we showed that replication of HBV in another human hepatoma cell line was not suppressed by IFN-lambda. However, these cell lines showed a similar level of transcription for the heterodimeric IFN-lambda receptor and the IFN-lambda-induced antiviral proteins. Our results suggest that the antiviral activity of IFN-lambda against HBV may be limited in human cells.