The thermolabile large T-antigen, encoded by the simian virus 40 early region mutant tsA58, was used to establish clonal cell lines (BMM-UV) from primary bovine myoepithelial cells. The BMM-UV cells have undergone more than 300 population doublings without any signs of senescence, and they contain the intranuclear large T antigen. At low confluency, they grow in a spindlelike manner and develop very long projections that most likely allow for communication of cells at a distance from each other. Establishment results in a decrease in the number of cells that contract in response to oxytocin compared with the parental nontransfected cells (20% versus 45%). Oxytocin responsiveness of BMM-UV cells increases when the cells are cultured in a medium supplemented with staphylococcal proteases. Proliferation of BMM-UV cells increases when they are cultured in the presence of epidermal growth factor (10 ng/ml) or insulinlike growth factor I (50 ng/ml). The BMM-UV cells may become a useful model to study growth properties, cell-to-cell communication, and the function of bovine mammary myoepithelial cells.