The antigen from a non-A, non-B antigen-antibody system previously described was purified and, when tested by immunodiffusion, was shown to produce patterns of identity with all antigen-positive sera of the system. The sedimentation coefficient S20,w of the antigen was estimated by rate zonal ultracentrifugation to be 48. Isopycnic ultracentrifugation in CsCl gradient of non-A, non-B antigen generated a single sedimentation peak at the density of 1.28. SDS-polyacrylamide-agarose gel electrophoresis showed that I125-labelled antigen migrated as a single band (molecular weight 3.0 X 10(6)); in addition, labelled material also migrated to the albumin region. With SDS-PAGE under reducing conditions, the antigen was shown to consist of seven polypeptides. Spherical particles of 23 nm in diameter, demonstrated by electron microscopy, could be aggregated by purified non-A, non-B antibody. They probably represent the antigen and not a complete viral particle. The characteristics of this antigen associated with non-A, non-B hepatitis appear therefore to be close to those of hepatitis B surface antigen.