The polymerase chain reaction (PCR) technique has been utilized for the detection of hepatitis B virus (HBV) DNA, and several factors related to the selection of primer pairs for the PCR amplification have been demonstrated. The sensitivity of the PCR assay was compared with that of slot-blot hybridization for detecting HBV-DNA. Analysis by the PCR technique with Southern blot hybridization provided a > 104-fold increase in sensitivity over the slot-blot hybridization analysis. Also, a rapid and sensitive PCR method for the detection of serum HBV-DNA was developed: HBV-DNA is released from virions by incubating serum with NaOH followed by neutral-ization with HCI. HBV-DNA sequences are then detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with successive sets of primer pairs. In testing serial samples from chimpanzees experimentally infected with HBV, HBV-DNA was detected 2-3 wk before the appearance of hepatitis surface antigen (HBsAg) and continued to be detectable for a short period after the production of antibody to HBsAg. Results from testing of human serum demonstrated that the majority of patients with HBsAg in serum had HBV-DNA as well and that some patients had HBV-DNA in serum in the absence of HBsAg.