Hepatitis B surface antigen (HBsAg) has been used for the detection of hepatitis B virus (HBV). Recently, HBV detection using the polymerase chain reaction (PCR) has been shown to be a direct measure of complete virions and to be potentially a very sensitive method. Therefore, we attempted to analyze the relationship between HBsAg detection and HBV DNA assay by PCR. We tested HBV DNA by a modification of the PCR technique in serial sera from five chimpanzees experimentally infected with HBV and 29 human sera. Our new method of PCR analysis is as sensitive as former methods but is more simple and rapid than existing procedures. In chimpanzee studies, HBV DNA was detected 2-3 weeks before the appearance of HBsAg, and continued to be detectable 2 weeks after the production of antibody to HBsAg. Also, 3 of 11 chronic hepatitis B patients who lost HBsAg were positive for HBV DNA in serum, while all of patients who recovered from acute viral infection were negative for HBV DNA in serum. These results indicate that HBV DNA detection by PCR analysis is the most sensitive method currently available to detect the presence of complete virions in serum.