Mutations of the p53 gene are the most frequent genetic lesion in breast cancer. Here, we examined p53 expression in a unique in vitro model of tumor progression derived from a single breast cancer patient (21T series). While the normal mammary epithelial, fibroblast and mesothelial cells derived from this patient expressed easily detectable functional p53 protein, the primary as well as metastatic tumor cell lines demonstrated a lack of p53 protein synthesis. 21T tumor cells failed to exhibit G1 cell cycle arrest upon exposure to gamma-irradiation, and their growth was suppressed by transfection of a normal p53 cDNA, demonstrating a lack of p53-mediated function in these cells. No p53 gene deletion or rearrangements were detectable. PCR and sequence analysis of the entire coding region of p53 gene revealed a novel mutation, an insertion of a single T within codon 33, which resulted in a frame-shift and early termination. The same mutation was observed in all 21T tumor cell lines. These results demonstrate a tumor cell-specific loss of p53 protein due to a frame-shift mutation, and suggest that p53 loss may occur at a relatively early step in breast tumorigenesis before metastatic seeding or emergence of tumor heterogeneity. In addition, the availability of normal and tumor-derived epithelial cells with known p53 sequences from a single breast cancer patient should facilitate understanding of the p53 regulation in mammary cells.