Serum samples from 62 women, inadvertently infected with hepatitis B virus in an in vitro fertilization program, were tested for the presence of hepatitis B virus-DNA using the polymerase chain reaction. Under conditions of a strict spatial separation of DNA extraction, amplification and product analysis, we succeeded in detection of as few as 360 hepatitis B virus particles per milliliter. Hepatitis B virus-DNA was detected with a high frequency during HBsAg and HBeAg antigenemia (98.5%) but also in the convalescent phase after appearance of antibody to HBsAg (18.2%). However, all patients with hepatitis B virus-DNA in convalescent sera were hepatitis B virus-DNA negative 3 to 6 mo later. All patients with HBeAg-positive samples showed hepatitis B virus-DNA positivity by polymerase chain reaction. For acute hepatitis, gene amplification restores the relationship between HBeAg and hepatitis B virus-DNA observed in serum from chronic hepatitis B patients and calls attention to the prolonged presence of hepatitis B virus-DNA in serum after generally accepted criteria for resolution of the infection have been reached.