Biophysical and biochemical analysis of hepatitis A virus has shown it to be a 27- to 32-nm icosahedral particle with 32 capsomers. The mature virion has a buoyant density of 1.33–1.34 g/cm3, a sedimentation coefficient of 156–160S, and is composed of four polypeptides with molecular weights of 30,000–33,000 (VPl), 24,000–27,000 (VP2), 21,000–23,000 (VP3), and 7,000–14,000 (VP4). The genome of hepatitis A virus consists of a single piece of single-stranded RNA which sediments at 32–35S and has a buoyant density of 1.64 g/cm3. The molecular weight of RNA is 2.25 × 106 when measured under nondenaturing conditions and 2.8 × 106 when measured under fully denaturing conditions. The genome contains a 40–80 nucleotide sequence of polyadenylic acid and is capable of infecting cell cultures. These findings, together with the observation that the virion is stable at pH 3.0 and resistant to ether and a temperature of 60° for 1 h, indicate that hepatitis A virus should now be classified as an Entero-virus within the family Picornaviridae.