Hepatitis A virus was purified fecal specimens obtained from 2 patients with naturally acquired hepatitis A. The purification procedure involved differential centrifugation, organic solvent extraction, agarose gel filtration, ion-exchange chromatography, and isopycnic ultracentrifugation in cesium chloride. Using immune electron microscopy and discontinuous SDS-PAGE, this procedure was found to be effective in removing extraneous material from hepatitis A virus. There was significant recovery of virus as judged by immune electron microscopy and solid-phase radioimmunoassay. Using this protocol, it has been possible to obtain virus preparations of sufficient purity and high enough titer to enable biochemical studies to proceed.