In chimpanzee hepatitis B virus (HBV) carriers, the mechanism of viral persistence has been examined by analyzing viral DNA molecules in liver and serum. Chimpanzee liver DNA contained two extrachromosomal HBV DNA molecules migrating on hybridization blots at 4.0 kb and 2.3 kb. There was no evidence for integration of HBV DNA into the host genome. The extrachromosomal molecules were distinct from Dane particle DNA and were converted to linear 3.25 kb full-length double-stranded HBV DNA on digestion with Eco RI. Nucleases S1 and Bal 31 converted "2.3 kb" HBV DNA to 3.25 kb via an intermediate of "4.0 kb" apparent length. The HBV DNA molecule that migrated at 2.3 kb represents a supercoiled form I of the HBV genome, and the molecule that migrated at 4.0 kb represents a full-length "nicked," relaxed circular form II. Evidence for supercoiled HBV DNA in serum Dane particles was obtained by production of form II molecules upon digestion with nuclease S1 or Bal 31. It is proposed that most Dane particles represent interfering noninfectious virus containing partially double-stranded DNA circles and that particles containing supercoiled HBV DNA may represent infectious hepatitis B virus.