We have defined a human breast tumor associated antigen using a murine monoclonal antibody (MAb DF3) prepared against a membrane-enriched fraction of a human breast carcinoma. This antigen has a MW of 290 kD and is detectable on the cell surface of human breast carcinoma cells using a live cell radioimmunoassay and fluorescence flow cytometry. More important, immunoperoxidase staining with MAb DF3 clearly distinguishes malignant and benign breast lesions. A cytoplasmic staining pattern has been observed with 40 of 51 (78%) breast carcinomas, but only one of 13 fibroadenoma or fibrocystic disease specimens. In contrast, reactivity of benign breast lesions with MAb DF3 primarily occurs along apical borders on ductules. These results demonstrate that the DF3 antigen is present on apical borders of more differentiated secretory mammary epithelial cells and in the cytosol of less differentiated cells.