The spontaneous production of elongated derivatives by cuboidal rat mammary epithelial cells was examined with the use of a series of single-cell clones grown in tissue culture. Four representative cell lines derived from a 7,12-dimethylbenz[a]anthracene-induced mammary tumor in an inbred WF rat were examined for morphologic stability, chromosome number, presence of immunoreactive fibronectin, laminin, prekeratin, and milk fat globule membrane (MFGM) antigens, ultrastructural characteristics, and tumorigenicity in syngeneic hosts. Conversion of cuboidal to elongated cells occurred by way of apparent morphologic intermediates, examples of which were isolated and cloned. Levels of immunoreactive fibronectin and laminin were greater in the elongated than the cuboidal clones, whereas the converse was true of prekeratin. MFGM antigens were present to a variable extent in all 4 clones. When grown on 0.3% collagen gels, cells of Rama 37 CL-A3 and Rama 37CS-A2 cuboidal clones exhibited surface microvilli and desmosomes. A minority of elongated cells contained microfilamental structures and pinocytotic vesicles similar to those seen in myoepithelial cells; the remainder lacked distinguishing ultrastructural features. After injection into syngeneic recipients, Rama 37 CL-A3 cuboidal line gave rise to glandular tumors consisting of cuboidal cells arranged in acinar structures, Rama 37 E5 elongated line induced spindle cell tumors, and Rama 37 CS-A2 and Rama 37 E8 lines induced tumors containing nests of mixed spindle and cuboidal cells. The majority of these tumors failed to metastasize.