An enzyme-linked immunosorbent assay for an antigen (termed DS-Ag) related to non-A, non-B hepatitis has recently been developed in our laboratory. The DS-Ag was derived from a proven infectious serum obtained from a patient with haemophilia and it was also detected in the acute phase serum of an experimentally infected chimpanzee. The DS-Ag has now been shown to have a buoyant density in CsCl of 1.32 g/cm2 and a sedimentation coefficient of 20 S. The mean molecular weight, determined by gel chromatography on 4% agarose columns, was found to be 0.9 x 10(6). The DS-Ag recovered from acute-phase chimpanzee serum has identical characteristics. The DS-Ag is stable for 1 h at 56 degrees C, at pH 3, in high concentration of CsCl or Nal and in 20% ether. Complete inactivation occurred after 1 min at 98 degrees C, at pH 2, in chloroform (1:1 mixture), in 1 ppm chlorine and in formalin (1:4000, 72 h 37 degrees C). Our studies showed that DS-Ag bears no resemblance to particles related to well-documented forms of viral hepatitis types A and B or other known pathogens. The DS-Ag is a complex substance which forms an antigenic entity related to non-A, non-B hepatitis.