In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV‐DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV‐DNA molecule which migrated at 4.0 kb position represents a full‐length “nicked,” relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV‐DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV‐DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence arc discussed.