Epitheliosis is a benign intraluminal proliferation in the breast ducts and lobules that needs to be distinguished from ductal carcinoma in situ (DCIS). The histogenesis and differentiation of cells comprising epitheliosis have been the subject of some controversy. We evaluated the expression of a high-molecular-weight keratin (34 beta E12), muscle-specific actin (HHF-35), and S-100 protein immunoreactivity in formalin-fixed sections of the breast with epitheliosis and DCIS. In 28 of 30 cases of epitheliosis, there was strong HMW keratin immunoreactivity in the streaming sheetlike intraluminal proliferations. In contrast, 35 of 40 cases of DCIS (nonpapillary and papillary) were nonreactive for HMW keratin; the other five were weakly reactive. Furthermore, in 10 cases of DCIS, some ducts had isolated or small aggregates of HMW keratin-positive benign cells on the luminal aspects of the neoplastic proliferation that were reminiscent of a pagetoid pattern. Muscle actin-stained sections were analyzed to assess myoepithelial (ME) cell participation in epitheliosis. Muscle actin-positive ME cells were present at the periphery of the involved ducts but were absent or rare within epitheliosis. The distribution of ME cells--i.e., at the periphery of the spaces involved--was similar in DCIS and epitheliosis. S-100 protein was weakly but relatively consistently expressed by epitheliosis, but all cases of DCIS were negative. Six cases of atypical ductal hyperplasia included in the study were negative for HMW keratin, muscle actin, and S-100 protein. The immunohistochemical profile of epitheliosis indicates that it is primarily an epithelial proliferation with strong HMW keratin and weak S-100 protein expression but without ME cell participation. The distinct differences in HMW keratin expression of epitheliosis and intraductal carcinoma appear to reflect a consistent antigenic difference in these two biologically distinct forms of proliferation.