Although alterations in the p53 tumor suppressor gene are detected frequently in human breast cancers, mammary tumors are observed infrequently in p53 null mice. This has led to the suggestion that absence of p53 alone is not sufficient for induction of mammary tumors. However, early death of p53 null mice from thymic lymphomas may obscure tumor phenotypes that would develop later. Therefore, p53 null mammary epithelium was transplanted into cleared mammary fat pads of wild type p53 BALB/c hosts to allow long-term analysis of mammary tumor phenotypes. Five treatments were compared for their effects on tumor incidence in hosts bearing transplants of p53 null and p53 wt mammary epithelium. The treatment groups were:(1) untreated;(2) continuous hormone stimulation with pituitary isografts;(3) multiple pregnancies;(4) DMBA alone; and (5) DMBA+ pituitary isografts. The tumor incidences in p53 null vs p53 wt mammary transplants for each treatment group were 62% vs 0%, 100% vs 0%, 68% vs 0%, 60% vs 4% and 91% vs 14%, respectively. The mammary tumors that developed in the p53 null mammary epithelium were all adenocarcinomas and were frequently aneuploid. These data demonstrate that the absence of p53 is sufficient to cause development of mammary tumors and that hormonal stimulation enhances the tumorigenicity of p53 null mammary epithelium to a greater extent than DMBA exposure alone. This model provides an in situ approach to examine the molecular basis for the role of p53 in the regulation of mammary tumorigenesis.