We have previously described the mortal human breast epithelial culture MCF‐10M, that was derived from fibrocystic breast tissue, was cultivated in medium with low calcium content for over 2 years, and spontaneously gave rise to the immortal MCF‐10 cell line. The emergence of immortalized cells, characterized by growth in conventional calcium levels, from mortal cells has proven to be a reproducible event. Here we report the establishment of a second immortal line from MCF‐10M, designated MCF‐10‐2, and establishment of the MCF‐12 immortal line after long‐term cultivation of MCF‐12M mortal cells from reduction mammoplasty tissue. DNA fingerprinting demonstrated the independent, human origin and lineage of the MCF‐10‐2 and MCF‐12 cell lines. Both lines require cortisol and EGF for maximal growth. The expression in these cultures of in vivo breast epithelial phenotypes was analyzed using 2‐dimensional gel Western blots and immunoper‐ oxidase staining with antibodies to cytokeratins and polymorphic epithelial mucin. MCF‐10M and MCF‐12M retain the cytokeratin profile of the luminal cell (7, 8, 18, 19), and also express cytokeratin 14, found predominantly in basal cells. The immortal lines express a similar profile, except that cytokeratin 19, a component of the fully differentiated luminal cell, is not expressed in the more uniform population seen in MCF‐10 and MCF‐12, but is retained in the morphologically mixed, less‐ selected population of MCF‐10‐2. Epitopes on the polymorphic epithelial mucin, recognized by antibodies HMFG 1, HMFG 2 and SM‐3, were detected in the mortal cultures and in the immortal lines, indicating the occurrence of both normal and abnormal mucin processing. MCF‐10, MCF‐10‐2 and MCF‐12 cells do not form tumors in nude mice, but appear to organize as duct‐like structures before regressing in the 5th week post injection.