MCF10AT cells are human breast epithelial cells which are able to establish preneoplastic lesions in immune deficient mice. Although, the preneoplastic phenotype was observed following transfection with a mutated c-Ha-ras (codon 12 valine), clones of MCF10AT are unable to form lesions in vivo. Restriction size fragment analysis was used to confirm that a clone unable to form the preneoplastic lesions retained the activated c-HA-ras and confirmed that the insertion site of the activated c-Ha-ras was the same for the clone as for MCF10AT1 which was selected for its ability to form lesions in vivo. Western blotting with antibody specific for the codon 12 valine c-Ha-ras demonstrated that p21 protein was comparable as well. Thus, the activated c-Ha-ras is not sufficient for the preneoplastic phenotype of human breast stem cell line MCF10AT.