An IgG3 monoclonal antibody (CU-1) directed to the Tn glycoprotein antigen, which does not cross-react with blood group A antigen, has been established after immunization of mice with a purified Tn antigen, followed by selection of hybridomas secreting antibodies with the desired specificity. The antibody binding with Tn antigen was inhibited by monosaccharide N-acetylgalactosamine (a mixture of alpha and beta anomers) and specifically by p-nitrophenyl-alpha-D-N-acetylgalactosaminide but not by p-nitrophenyl-beta-D-N-acetylgalactosaminide, and the antibody binding was competitively inhibited by an IgM anti-Tn antibody NCC-LU-81. The antibody was not reactive with blood group A antigen, in contrast to previously reported anti-Tn antibodies which showed various degrees of cross-reactivity. Immunoperoxidase staining of various tumors and normal tissues with CU-1 showed a strong preferential or specific staining of sections from various cancer tissues over normal counterpart tissues, except for goblet cells in colon and some mammary gland epithelia. The positive staining in cancer and normal tissues was independent of the blood group ABH status of the host. Among various human cell lines tested, a number of cancer cell lines showed various degrees of antibody binding activity, although the binding did not correlate with the pathohistological type of the tumor cell lines. The intensity of antibody binding to cells was not proportional to the susceptibility of cells to the antibody-dependent cytotoxicity.