Monoclonal antibody 7B10, raised against the human breast cancer cell line T47D, identifies an antigen found in human breast carcinomas and in normal breast. Western blot and immunoprecipitation studies detected a Mr 76,000 antigen in cytosol, cell membrane, and cell culture supernatants of T47D cells. 7B10 binding to T47D cell extracts was affected by proteolytic digestion with protease type VI, trypsin, and subtilisin while it was not altered by neuraminidase digestion. Adsorption of breast cancer cell line extracts with concanavalin A reduced 7B10 immunoreactivity more than 70%. These results suggest that the antigen is a glycoprotein and that the epitope does not contain sialic acid. 7B10 was reactive with neither human milk fat globule membrane, nor skimmed milk, nor the milk-derived HBL 100 cell line. Conversely binding was detected in more than 50% of normal breast epithelial cells in culture. 7B10 immunostaining was positive on frozen sections of normal breast and nonmalignant mastopathies in 30 to 90% cells. In frozen sections of other normal tissues, 7B10 immunoreactivity was detected only in colon, apocrine glands of skin, parotid ducts, and luteal phase endometrium, confirming previous data on paraffin sections. Strong, homogeneous immunostaining was observed on frozen sections of intraductal and invasive lobular breast carcinomas (100% of cases), while more heterogeneous staining was found on invasive ductal carcinomas. Colon and rectal carcinomas, one carcinoma of the esophagus, and some cells in serous ovarian carcinomas also showed 7B10 reactivity. Immunoblotting of the 7B10-immunoreactive fraction isolated by Sepharose CL-6B chromatography of a breast carcinoma tissue sample extract identified the Mr 76,000 antigen, which was also detected in several breast cancer specimens, in colon adenocarcinomas, and in serous ovarian carcinoma fresh tumor extracts. The Mr 76,000 glycoprotein described here represents a breast cancer-associated antigen previously undescribed, mainly expressed in normal breast and breast tumors.