Dilutions of human sera containing between 103 and 105 chimpanzee infectious doses of hepatitis B virus per ml, subtype adr or ayw, were treated with either 1 m̈g/ml pepsin at pH 2.0 for 18 hours, 8 M urea for four hours, or 1:4,000 formalin for 72 hours. One ml of the serum containing hepatitis B virus subjected to each of the procedures was inoculated intravenously into one or two susceptible chimpanzees (total of eight chimpanzees). No evidence of hepatitis B infection was detected in weekly serum samples from the chimpanzees during six months of observation. These three procedures are currently applied during manufacture to inactivate HBV which might be present in the hepatitis B vaccine licensed in the United States. The data from this study combined with data documenting that the physical purification of the vaccine is capable of removing hepatitis B virus provide assurance that there is no residual live hepatitis B virus in the vaccine.