Abstract The aim of this study was to investigate the retention in culture of the antigens characteristic of the two mammary epithelial subclasses, basal and luminal epithelium. Primary and secondary cultures of normal human mammary-gland cells were used for immunolocalization experiments with monoclonal antibodies to luminal and basal epithelium. In contrast to the in vivo situation, in which reactivity was only seen in basal cells that were negative for the luminal antigen, we found the homogeneous expression of the basal marker by all of the cultured cells at second passage, and the simultaneous expression of the luminal marker by some of these cells. Characterization of the basal antigen expressed in culture using sodium-dodecyl-sulfate/polyacrylamide gel electrophoresis and immunoblotting techniques showed it to be a 51-kilodalton keratin peptide with an isoelectric pH of 5.4, and confirmed its similarity to the antigen expressed in vivo. Our findings thus demonstrated the coordinate expression of the basal and luminal antigens in cells cultured on solid substrates. The availability of monoclonal antibodies to epithelial-subclass-specific markers of the human mammary gland now makes it feasible to search for culture conditions that would allow the maintenance and manipulation of cell differentiation in vitro.