Recent observations in children with rotavirus gastroenteritis and in infant mice given rotavirus vaccine by oral administration suggest that this well-known gastrointestinal pathogen may infect the liver. To examine this possibility, the susceptibility of Hep G2 cells to infection with a variety of rotavirus strains was tested. These cells were used because they are considered to be well differentiated and exhibit many liver-specific functions. The Hep G2 cells supported the growth of the simian strain rhesus rotavirus (MMU 18006), a strain currently being used in vaccine trials, but did not support the growth of any human strain (D, DS1, Price or ST3). The rhesus rotavirus infection was cytopathic and resulted in release of lactate dehydrogenase. Rhesus rotavirus growth in Hep G2 cells displayed trypsin-enhanced infectivity and was inhibited by pretreatment of cells with Arthrobacter ureafaciens neuraminidase but not with neuraminidase from Clostridium perfringens. Hep G2 cells were also permissive for another simian strain (SA11), a bovine strain (UK) and single gene substitution reassortants containing VP7 (the major outer capsid neutralization protein) from a human rotavirus strain and the remaining 10 genes from either rhesus rotavirus or UK. In general, UK and its reassortants produced lower levels of antigen than did rhesus rotavirus and its reassortants. Hep G2 cells and other hepatic cell lines may prove to be useful tools to explore the hepatotropic potential of wild-type rotaviruses and candidate vaccine strains. (HEPATOLOGY 1990;12:638–643).