A 10(-3) dilution of pooled serum (positive for hepatitis B e antigen and DNA polymerase activity) containing hepatitis B virus (HBV) in a titer 10(5) times the chimpanzee-infectious dose, was heated under water maintained at 60 C for 10 hr. There was a twofold decrease in the titer of hepatitis B surface antigen (HBsAg) as measured by reverse passive hemagglutination after the heat treatment. The heated, diluted serum was still infectious and caused HBV infections in both seronegative chimpanzees given 1-ml iv inoculations of the diluted serum. However, the infectivity of the virus was decreased approximately 10(4)-fold by heat treatment as judged from the prolonged incubation period before appearance of HBsAg in blood. This figure was based on the inverse linear relation between the dose of HBV and the incubation period. The incomplete inactivation of HBV by heat treatment at 60 C for 10 hr should be emphasized because it is widely accepted that heat treatment destroys HBV.