Embryonic antigen associated with an aminoazo dye-induced rat hepatoma was identified in the serum from rats bearing progressively growing tumours. Antigenic activity in serum samples was detected by their capacity to neutralize multiparous rat serum antibody reacting with surface embryonic antigens expressed upon viable hepatoma cells as assessed with use of the indirect membrane immunofluorescence test. Serum taken at various stages of tumour growth from hepatoma-bearing rats was separated by Sephadex G-150 gel filtration column chromatography at pH 7.3 and pH 2.8 with use of procedures designed to identify free circulating antigen and antigen derived from immune complexes. Hepatoma-associated embryonic antigen was demonstrable in tumour-bearer serum in a free form most markedly in the later stages after implantation of tumour cells (from the end of the 2nd week to the 5th week of tumour growth). Antigenic activity in fractions derived from immune complexes was detected earlier during tumour development (from day 8 after tumour induction), and this was present in all serum samples taken up to the 5th week after tumour cell inoculation.