We developed a polymerase chain reaction assay for the direct detection of hepatitis B virus in paraffinembedded liver tissue and applied this assay to determine whether hepatitis B virus DNA exists in livers with chronic hepatitis non-A, non-B. Fifty five liver biopsy samples were studied: 11 from patients with HBeAg-positive chronic hepatitis (paraffin-embedded) and 44 from patients with chronic hepatitis non-A, non-B (21 paraffin-embedded 25 fresh frozen). Thirty three (75%) of the non-A, non-B cases were positive for hepatitis C virus antibodies. Approximately 1 to 10 ng of DNA was extracted from the paraffin-embedded tissue and amplified using oligonucleotide (23-mer) primers specific for the S gene (positions 261 to 692). The β-globin gene was used as an internal control for sensitivity because this is a single copy gene and allows for relative quantification. In each of the chronic hepatitis B livers, the expected 432-base-pair amplification product for hepatitis B virus DNA and β-globin gene product were both detected. On the other hand, in the 21 paraffin-embedded chronic hepatitis non-A, non-B livers, no hepatitis B virus DNA was detected, although β-globin gene was observed in all. Furthermore, in all 25 frozen non-A, non-B livers, β-globin gene was observed, but no hepatitis B virus band was seen. The limit of detection of hepatitis B virus DNA by this method was estimated to be one genomic copy of hepatitis B virus DNA per cell. Thus all patients with chronic hepatitis B, but no patients with clinically diagnosed non-A, non-B hepatitis, had hepatitis B virus DNA in liver at a level of or above one copy per hepatocyte. (HEPATOLOGY 1991;13:167–171).