An enzyme-linked immunosorbent assay (ELISA) was developed, based on sera from patients convalescent from non-A, non-B hepatitis and haemophilia A patients. Using this ELISA an antigen (DS-Ag) was detected which appears to be related to non-A, non-B hepatitis. The serum of another patient with haemophilia A, which was strongly positive for the DS-Ag, caused non-A, non-B hepatitis in a chimpanzee. DS-Ag could be detected in the serum of the chimpanzee during the acute phase of the infection. The ELISA for DS-Ag did not react with either hepatitis A or B virus antigens, or with Epstein-Barr virus or Cytomegalovirus. The assay was provisionally evaluated using sera from different groups of patients. From 17 patients with post-transfusion hepatitis non-A, non-B, 11 were found positive in the ELISA for DS-Ag (65%). A relatively high prevalence of DS-Ag (17%) and -antibodies (20%) was found in patients with haemophilia, who are regularly treated with blood clotting factor concentrates. The DS-Ag was found in 8 of 1400 (0,6%) volunteer blood donors, and antibody to DS-Ag in 3% of the sera. Remarkably, a high incidence (41%) of antibodies to DS-Ag was found in prostitutes, suggesting that this antigen may be transmitted by a sexual route as well. The DS-Ag was pelleted by ultracentrifugation for 4 h at 100.000 xg and was found to have a buoyant density of 1.32 g/cm3 in a CsCl gradient.