Rapidly replicating, cytopathic (rr/cpe+) variants of hepatitis A virus (HAV) isolated from persistently infected BS-C-1 cells have numerous mutations from cell culture-adapted rr/cpe- HAV. To determine which mutations in one rr/cpe+ virus, HM175/18f, determine enhanced replication in BS-C-1 cells, a series of chimeric viruses was rescued from infectious cDNAs in which HM175/18f genomic segments were placed within the background of a related rr/cpe- virus, HAV/7. Chimeric viruses containing the P2 region of HM175/18f produced replication foci in BS-C-1 cells that were larger than HAV/7, but not as large as HM175/18f virus. Enhanced viral replication required mutations in both 2B and 2C proteins, suggesting that these proteins remain closely associated during replication. Mutations in 5' nontranslated RNA (5'NTR) or P3 proteins had no independent effect, but acted cooperatively with mutations in P2 proteins to enhance replication and render the virus capable of conventional plaque formation. Cytopathic effects correlated with viral replication capacity and were not the result of any single mutation. Full expression of the rr/cpe+ phenotype required mutations within the 5'NTR, P2, and P3 segments. These results suggest novel interactions between the 5'NTR and P2 proteins during HAV replication and provide useful new infectious cDNA clones.