Antibody-based cancer immunotherapy relies on the identification and characterization of target antigens and the development of potent antibodies recognizing the target. Here we report the expression analysis and molecular characterization of the differentiation antigen NY-BR-1, which we previously identified by using the SEREX (serological analysis of recombinant cDNA expression libraries) method. Corroborating methodologies, including mRNA quantitation and immunoblotting show that NY-BR-1 is strongly expressed in >70% of 129 breast tumors. Application of a NY-BR-1 specific antibody demonstrated NY-BR-1 expression in primary and metastastic breast cancers. In contrast, most of the breast cancer cell lines tested, expressed only low NY-BR-1 levels. Importantly, confocal microscopy revealed that ectopically expressed NY-BR-1 localizes to the cytoplasm and the cell membrane. NY-BR-1 localization in breast cancer specimens was also confirmed by immunohistochemistry. Bioinformatic analysis and deletion mutagenesis further show that NY-BR-1 membrane localization is mediated by 2 cis-active membrane targeting domains. Biochemical surface labeling and FACS analysis of live cells further characterize NY-BR-1 as a transmembrane protein, which can be specifically recognized by the anti-NY-BR-1 antibody on the surface of vital cells. The strong expression of NY-BR-1 in breast tumors, its cytoplasmic and membrane localization and accessibility to an ectopically applied antibody now suggest to pursue NY-BR-1 as a potential target for antibody-based therapies in breast cancer patients.