Polymerase chain reaction (PCR) was used to detect hepatitis B virus DNA in the sera and livers of nine patients with chronic hepatitis B after treatment-induced or spontaneous loss of serum hepatitis B surface antigen. Patients were evaluated at intervals ranging from 3 to 67 months after disappearance of hepatitis B surface antigen. PCR was performed using primer pairs from the surface and core gene regions, and surface gene products were quantitated. Liver tissue was also evaluated by in situ hybridization to assess viral transcription. Five of the nine patients had viral DNA detectable in serum by PCR. Quantitation of polymerase chain reaction products in serum and liver showed that the DNA levels tended to decline progressively after antiviral therapy. Six of seven surface antigen-negative patients tested had detectable viral DNA in the liver, and four of the six DNA-positive patients were negative for DNA in serum by PCR. None had surface gene messenger RNA. Thus, it is concluded that hepatitis B virus DNA may be detectable by PCR in liver tissue years after the disappearance of hepatitis B surface antigen, even in the absence of detectable hepatitis B virus DNA in serum.