Preneoplastic lesions in the mammary gland represent a population of cells at increased risk of progression to tumors. Because p53 is the most commonly mutated gene in human breast cancer, we sought to determine whether mutations in p53 were present in preneoplastic lesions or were acquired during progression to overt tumors. In the mouse mammary gland, hyperplastic alveolar nodules (HAN) are the most common preneoplastic lesion. Analysis of the TM series of transplantable murine HAN outgrowths and tumors allowed the status of p53 to be determined at distinct stages of mammary tumorigenesis. Alterations in the p53 gene or in the pattern of p53 protein expression were observed in all five HAN outgrowth lines examined. Altered expression of p53 protein was detected in 3 of 5 TM HAN outgrowth lines as determined by immunohistochemistry. Overexpression of nuclear p53 was detected in only a fraction of the cells (10–50%) in TM3 and TM4 HAN outgrowths, whereas in tumors that arose from TM4 HAN outgrowths, the proportion of cells overexpressing p53 protein approached ∼100%. Despite overexpression of p53 in TM3 HAN outgrowths, no tumors have developed in this line. The TM9 outgrowth line exhibited a different pattern of p53 expression by immunohistochemistry: p53 protein was overexpressed in the cytoplasm of virtually all cells in the HAN outgrowths but was localized to the nuclei of TM9 tumor cells. Direct sequencing of p53 transcripts from tumors and cell lines revealed various genetic changes: point mutations in exons 4 and 5 (TM2H, nonsense; TM4, missense); a deletion in exon 5 (TM4); and an insertion in exon 7 (TM3). Although p53 protein was overexpressed in TM9 tumors, it was shown to be wild-type both by immunoprecipitation and direct sequencing of the entire coding region of the cDNA. TM4 cells were homogeneous with respect to mutant p53 genotype and uniformly expressed p53 by immunohistochemical staining in vitro, but transplantation of TM4 cells to fat pads of BALB/c hosts resulted in HAN outgrowths in situ in which <50% of the cells expressed the mutant p53 at detectable levels. In summary, mutation of the p53 gene and overexpression of p53 protein can occur in preneoplastic mammary epithelial cells, and those mutations are maintained in tumors that arise from the HAN. It appears that mutation of p53 conferred a biologically relevant growth advantage to cells in vivo. Overexpression of p53 in one outgrowth line in the absence of mutations in the coding sequence suggests the presence of cellular factors that can enhance accumulation of wild-type p53 protein. Conversely, expression of mutant p53 was decreased when cells were grown in situ, implicating the presence of cellular factors that can suppress p53 expression in vivo. These observations demonstrate that the p53 pathway may be a common target for mutation in murine mammary tumorigenesis.