Hanganutziu-Deicher (H-D) antigen is classified as a heterophile antigen and chemically defined as a glycoconjugate which contains N-glycolylneuraminic acid. H-D antigens are absent from normal human tissues, but can be expressed on a variety of human malignant cells, including melanoma. Natural anti-H-D antibodies have been detected in man with and without malignancies, but in this study when the level of antibody was compared between healthy adults and patients with melanoma, elevated anti-H-D antibody levels were found more frequently in melanoma patients for both IgM (p = 0.0001) and IgG (p = 0.0001). The present study was designed to evaluate the significance of the H-D antigen-antibody system in melanoma suppression. Sera from melanoma patients containing anti-H-D antibody reacted strongly to H-D antigen expressed on melanoma by means of flow cytometry. In a complement-dependent cytotoxicity assay this antibody killed melanoma cells in vitro. In vivo significance of the antibody was assessed by evaluating the relationship between the antibody levels and the clinical course in patients with stage II melanoma. Antibody levels were measured by enzyme-linked immunosorbent assay using a H-D glycoprotein antigen isolated from bovine erythrocytes. A significantly higher level of IgG (p = 0.0640) and IgM (p = 0.0644) anti-H-D antibody was demonstrated in those patients who were free of disease more than 5 years after surgery than in those who relapsed within 2 years. This study provides a rational basis for immunotherapy targeting H-D antigen in human melanoma.