Studies were conducted on the preparation, inactivation, safety, and immunogenicity of a prototype hepatitis A virus vaccine prepared from infected cell cultures. BS-C-1 cells maintained in medium 199 without serum were infected with the HM175 strain of hepatitis A virus and harvested after 21-28 days. The harvested virus preparation contained 6.8-7.4 (log 10) cell culture infectious doses/ml. After exposure to 1:4,000 formalin at 35 C, the infectivity titer decreased 10(6)-fold in 30 hr at an exponential rate, although virus was detected in 5.0-ml vaccine samples for up to three days. Three separate vaccine lots elicited antibody in all the guinea pigs given three doses. Owl monkeys given three doses of vaccine did not have any evidence of HAV infection but developed antibodies identifiable by radioimmunoassay and serum neutralization tests. After either oral or intravenous challenge with at least 10(6) monkey infectious doses of a virulent field strain of hepatitis A virus, none of the vaccinated monkeys shed virus in their feces or had elevated serum levels of alanine aminotransferase. The findings suggest that an effective inactivated whole virus hepatitis A vaccine can be prepared from cell culture.