The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA.