The PIK3 signaling pathway has been identified as one of the most important and most frequently mutated pathways in breast cancer. Somatic mutations in the catalytic subunit of PIK3CA have been found in a significant fraction of breast carcinomas, and it has been proposed that mutant PIK3CA plays a role in tumor initiation. However, the majority of primary human tumors analyzed for genetic alterations in PIK3CA have been invasive breast carcinomas and the frequency of PIK3CA mutations in preinvasive lesions has not been explored. To investigate this, we sequenced exons 9 and 20 of PIK3CA in pure ductal carcinoma in situ (DCIS), DCIS adjacent to invasive carcinoma, and invasive ductal breast carcinomas. In a subset of cases, both in situ and invasive areas were analyzed from the same tumor. We found that the frequency of PIK3CA mutations was essentially the same (∼30%) in all three histologic groups. In some cases, in situ and invasive areas of the same tumor were discordant for PIK3CA status, and in two cases in which multiple invasive and adjacent in situ areas within the same tumor were analyzed independently, we detected intratumor heterogeneity for PIK3CA mutations. Our results suggest that mutation of PIK3CA is an early event in breast cancer that is more likely to play a role in breast tumor initiation than in invasive progression, although a potential role for exon 9 mutations in the progression of a subset of DCIS cases cannot be excluded. Cancer Res; 70(14); 5674-8. ©2010 AACR.