The virulence of a clonally isolated, cell culture-adapted hepatitis A virus (HM175/p16) was assessed in 4 seronegative owl monkeys inoculated intravenously with 2.8 x 10(4) radioimmunofocus-forming units of virus. The virus was highly attenuated, even though its complete nucleotide sequence contains only 19 mutations from the wild-type genome. Only 3 monkeys developed antibodies to hepatitis A virus (only 2 within 96 days of virus inoculation). One monkey had viremia and significantly elevated serum aminotransferase levels. In this animal, maximum viremia and fecal shedding of virus occurred 30-33 days after inoculation. In contrast, in earlier studies of a related cell culture-adapted but still hepatovirulent virus (HM175/S18), viremia was documented in 6 of 6 animals and peak viremia and fecal shedding of virus occurred 18 or 19 days after intravenous inoculation of about one-tenth as much virus.