The development and progression of human breast neoplasia is characterized by the accumulation of numerous somatic genetic alterations. Although mutation of the p53 tumor suppressor gene is one of the most common alterations identified in invasive carcinomas, it is not clear whether mutations usually occur in noninvasive lesions before the development of invasion. To investigate the presence and heterogeneity of p53 mutations in breast neoplasia, we studied a morphological spectrum of paired lesions including invasive carcinomas and adjacent noninvasive epithelial lesions. Using 18 invasive ductal carcinomas with known p53 mutations, tissue samples were microdissected from formalin-fixed, paraffin-embedded tissue sections, and mutations in exons 4-8 of the p53 gene were identified by PCR amplification, single-strand conformational polymorphism, and direct sequencing. Multiple geographically distinct foci of invasive carcinoma were microdissected from six different invasive carcinomas, and all samples from each case had the same mutation. The absence of mutation heterogeneity provides evidence that p53 mutations occurred at the time of, or before, the clonal selection of the dominant component of the invasive carcinoma. To delineate the timing of p53 inactivation further in these cases, histological slides were reviewed to identify all noninvasive epithelial lesions. There were eight cases with associated ductal carcinoma in situ (DCIS), and in total, 27 distinct tissue samples representing a spectrum of histological subtypes and grades of DCIS were microdissected. In all 27 samples, the identical p53 mutation was identified in the DCIS as was present in the invasive carcinoma. In contrast, no p53 mutations were identified in any of the 21 microdissected foci of epithelial hyperplasia analyzed, including one sample with atypia. Together, these findings provide support that p53 mutations commonly occur early in breast neoplasia, usually at the stage of DCIS, but are not often identified in foci of hyperplasia. These findings support an important biological role for p53 mutation in progression from noninvasive precursors in breast neoplasia and provide further evidence that p53 mutation could have potential use as a molecular marker.