The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D line. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of beta-casein mRNA in the presence or absence of prolactin. The heterogeneous COMMA-D line, but none of the clonal lines, was induced by the presence of prolactin to produce significantly increased levels of beta-casein MRNA. The inducibility of beta-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. Individual matrix components of laminin, fibronectin, heparan sulfate, heparan, or hyaluronic acid were not effective as substrata for the induction of beta-casein mRNA. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.