The nucleotide sequences of the VP1 coding region of two newly characterized, cell culture-adapted hepatitis A virus (HAV) strains (RG-SB11 and RG-SB16) were analyzed and compared with homologous regions of previously characterized HAV strains of human or monkey origin, and at different levels of tissue-culture adaptation. In particular, HM175wt and its derivative strains and MBB, LCDC1, PA21, and AGM27 isolates were considered. RG-SB11 and RG-SB16 HAV strains were derived from a pathogenic isolate from an acutely infected patient, purified from stool, and subjected to different strategies of adaptation. Several nucleotide differences were observed, but high conservation was found in the predicted VP1 protein sequences, which confirms structural constraints for this region. Furthermore, comparative amino-acid sequence analysis of VP1 from all HAV isolates studied has shown, particularly for those from naturally infected monkeys, that differences are limited to the amino and carboxy-terminal part of the molecule. The results of phylogenetic analysis have confirmed the common origin of the RG-SB11 and RG-SB16 strains. The complete nucleotide sequences of the VP1 coding region of the RG-SB11/16, HM175 derivative strains and of other HAV strains has shown that branch-length evolution can give a measure of the evolution of HAV during adaptation processes.