A tissue culture system is described which permits identification and propagation of isolates from the blood of hepatitis patients. Techniques first described in 1956 have been greatly improved. Viruses thus isolated produced hepatitis in 3 clinical studies. Incubation period, symptoms, clinical manifestations, and laboratory findings were typical of infectious hepatitis. Viruses were consistently reisolated from these cases and specific neutralizing antibodies were demonstrated. Limited studies indicated at least 3 distinct virus serotypes, 2 of which produced hepatitis with jaundice. Application of ultrafiltration, ultracentrifugation, and electron microscopy to infectious tissue culture fluid revealed 12 to 18 mμ particles not found in controls. Similar particles were noted in infected cultured cells. Despite difficulties, these clinically proved laboratory techniques insured continued characterization of hepatitis viruses. These techniques may eventually become tools for diagnosis and preparation of antigens and vaccines.