Biochemical properties of Epstein-Barr virus (EBV)-related soluble antigen in non-producer Raji Burkitt lymphoblastoid cells, assayed by the indirect single radial immunodiffusion (ISRD) test, were investigated. The soluble ISRD antigen retained activity even after exposure to a temperature of 80 degrees C. The antigen was precipitated in 40% saturated ammonium sulfate and ISRD activity was recovered from the precipitate when reconstituted into a solution. Isoelectric focusing and crossed immunoelectrophoretic analyses revealed that the esoelectric point of the ISRD antigen is pH 4.8 with an electrophoretic mobility similar to that of serum alpha-globulin. With Sephadex G-200 or Sepharose 6B gel filtration, ISRD activity was obtained as a single peak which corresponded to the activity absorbing anti-complement immunofluorescence. The molecular weight of the present EBV-related soluble antigen was estimated to be 220,000-2408000 daltons.