Hepatitis A virus was purified from fecal specimens obtained from 3 patients with naturally acquired hepatitis A, by a process of differential centrifugation, chloroform extraction, column chromatography, and isopycnic ultracentrifugation. Analysis of purified virus by discontinuous SDS-PAGE revealed three major polypeptides with molecular weights of 34,000, 25,500, and 23,000 daltons. These polypeptides appear to be specific for hepatitis A virus and have similar molecular weights to three of the four major polypeptides reported for members of the genus Enterovirus within the family Picornaviridae.