Rapidly replicating, cytopathic (rr/cpe+) variants of hepatitis A virus (HAV) isolated from persistently infected BS-C-1 cells have numerous mutations from the sequence of the cell culture-adapted HM175 strain HAV that served to initiate the persistent infection (Lemon et al., J. Virol. 65:2056-2065, 1991). To determine which of the mutations in one such rr/cpe+ virus, HM175/18f, are responsible for enhanced replication in BS-C-1 cells, a series of chimeric virus cDNAs was constructed in which HM175/18f virus genomic segments were placed within the background of an infectious cDNA of a related rr/cpe- virus, HAV/7 (HM175/P35). The replication capacity of chimeric viruses rescued from these cDNAs was assessed in BS-C-1 cells by the size of replication foci in radioimmunofocus assays. Chimeric viruses containing the mutated P2 region of HM175/18f virus produced replication foci that were larger than HAV/7 virus, but not as large as HM175/18f virus. This enhancement in replication required mutations in both the 2B and 2C protein coding regions, suggesting that these proteins act cooperatively and remain closely associated during replication. Mutations in the 5' nontranslated RNA (5'NTR) and P3 proteins had no independent effect on viral replication, but mutations in both of these genomic regions acted cooperatively with mutations in P2 proteins to enhance viral replication and render the virus capable of conventional plaque formation. Cytopathic effects associated with HAV replication are thus correlated with viral replication capacity, and do not appear to be the result of any single mutation. Full expression of the rr/cpe+ phenotype required mutations within the 5'NTR, P2 and P3 segments of the HM175/18f virus genome. These results suggest novel interactions between the 5'NTR and P2 proteins of the HAV genome during viral replication, and provide useful new infectious cDNA clones for further molecular studies of HAV.