The murine monoclonal antibody (MAb) designated DF3, reacts with a 300-kd human mammary epithelial antigen which is expressed on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. Human mammary tumors have been evaluated for the level of DF3 antigen as a correlate to clinicopathologic parameters related to degree of tumor differentiation: nuclear grade (NG), histologic grade (HG), and estrogen receptor status (ER). More DF3 antigen was present in breast carcinomas with NG 1 and 2 as compared to tumors with NG 3 (p = .002). Similarly DF3 antigen presence was greater in HG 1 and 2 tumors than in HG 3 (p less than .001). The results also demonstrate that quantitative differences in the presence of the DF3 differentiation antigen correlate with estrogen receptor status. Twenty-two of 23 ER positive tumors were also DF3 positive. Only 6 of 23 ER negative tumors were reactive to MAb DF3 (p less than .001). There was, however, no correlation between DF3 reactivity and absolute levels of estrogen or progesterone receptor. These findings confirm our hypothesis that MAb DF3 reacts to a differentiation antigen present in some human breast carcinomas. The DF3 antigen phenotype can serve as an independent phenotypic marker with correlations to standard indicators of degree of differentiation and estrogen receptor status of infiltrating ductal carcinomas of the breast, and should thus be evaluated as a prognostic indicator in breast cancer patients. The data also suggests that DF3 histochemistry may be a useful alternative in assessing estrogen receptor status of small breast cancers where there is an insufficient amount of tumor present for biochemical assay of hormone receptor levels.