An affinity of horseradish peroxidase for hepatitis B surface antigen (HBsAg), irrespective of antigen-antibody conjugation, was disclosed incidentally, and a new staining method of HBsAg was established. The new technique is simple, inexpensive and available for formalin-fixed, paraffin embedded sections, and does not require fresh liver tissue. Compared with three other methods, involving Shikata's orcein staining, the conventional immunoperoxidase method, and the peroxidase-anti-peroxidase method, it showed high specificity and sensitivity, which were not lower than the other three methods. Conjugation of horseradish peroxidase with HBsAg was confirmed by affinity chromatography. Attention should be paid to the fact that there is a possibility of obtaining false positive results by immunoperoxidase methods, staining antigens other than HBsAg in HBsAg positive liver tissue because of antigen-antibody independent affinity of peroxidase for HBsAg.