Breast ductal carcinoma in situ is an intraductal proliferation of malignant epithelial cells that diffuse within the ductal system without stromal invasion. Our finding that a subset of these tumors express CD31/platelet endothelial cell adhesion molecule-1 suggests that breast cancer represents an informative model for studying the involvement of the molecule in the morphogenesis, differentiation, and diffusion of this disease. Transfection of CD31 in MDA-MB-231 cells caused reduction in growth, loss of CD44, and acquisition of a ductal morphology. The same effects were maintained in vivo, in which CD31(+) tumors grew with in situ-like aspects, papillary differentiation, and a secretory phenotype. CD44 was down-modulated, with the CD31(+) cells blocked in the G(1) phase. The morphology was highly similar to what was observed in some human CD31(+) ductal carcinomas in situ. MDA-MB-231 mock cells grew in solid sheets, lacking stromal material, and displaying high levels of CD44 and proliferation. CD31(+) cells acquired motility characteristics in in vitro assays, a finding confirmed in vivo by the diffusion of human tumor cells throughout the normal ducts residual in the murine mammary gland. In conclusion, CD31 expression reverts the undifferentiated morphology and aggressive behavior of MDA-MB-231 cells, indicating its active role in the morphogenesis of breast ductal in situ carcinomas.