To determine the molecular changes associated with adaptation of hepatitis A virus (HAV) to growth in cell culture, the genome of a cell culture-adapted variant of HM175 strain HAV (p16 HM175, 16th in vitro passage level) was molecularly cloned and the complete nucleotide sequence of the virus was determined. Compared with wild-type virus, p16 HM175 replicates efficiently in monkey kidney (BS-C-1) cells (approximately 58 RNA-containing particles per one infectious unit, compared with 2.4 × 10⁵ for wild-type HM175). The nucleotide sequence of p16 HM 175 revealed a total of 19 mutations from the wild-type genome, including 5 mutations in the 5′ nontranslated region, 1 mutation in the 3′ nontranslated region, and 13 mutations predicting 8 changes in the amino acid sequences of HAV proteins. Only one amino acid substitution occurred among the capsid proteins (VP2), while others involved proteins 2A, 213, 2C, Vl3g, and 3Dpol. When the sequence of p16 virus was compared with that reported previously for an independently isolated, cell culture-adapted variant of HM175 virus (J. I. Cohen et al., (1987). Proc. Natl. Acad. Sci. USA 84, 2497–2501), there were three identical mutations in nontranslated regions of the RNA, and four mutations involving identical amino acids in proteins VP2, 2B, and 3Dpol. The distribution of these mutations within the genome suggests that changes in RNA replication may be of primary importance in adaptation of the virus to growth in vitro. These data are thus helpful in understanding the molecular basis of adaptation of HAV to cell culture and, since attenuation frequently accompanies adaptation of virus to growth in cell culture, may be of benefit in planning for attenuated vaccine development.