The MCF10AT premalignant human breast epithelial cells form benign ductal structures in immunodeficient mice which sporadically progress to carcinoma in situ and invasive cancers of different histologic types. MCF10CA1 cell lines are malignant variants derived by serially passing small pieces of tumors in athymic mice before establishing cells in culture. As these MCF10CA1 variants gave rise to heterogeneous tumors, some cell lines were cloned. Inoculated into immunodeficient mice, these variants produce squamous carcinomas with an undifferentiated component or adenocarcinomas also with an undifferentiated component. Immunohistochemistry utilized antibodies against DF3, c-erbB-2, cyclin Dl, m keratin, p keratin, p53, B72.3 and estrogen receptor. We detected characteristic patterns for squamous carcinomas, for adenocarcinomas, and for each undifferentiated component, that is the undifferentiated components of the squamous and glandular carcinomas were distinct. Only adenocarcinomas were focally ER positive. One uncloned variant that produced cancers with a glandular component, MCF10CA1h, was cloned and cells were injected into mice. This clone produced only undifferentiated carcinomas that, compared to tumors formed by the parental uncloned variant, had lost ER, DF3 and c-erbB-2 expression, but more strongly expressed p53. Our data demonstrate the potential of the premalignant MCF10AT model to generate heterogeneity, including both estrogen receptor-positive as well as estrogen receptor-negative tumors, during progression.