14-3-3 proteins comprise a family of highly conserved and broadly expressed multifunctional regulatory proteins that are involved in various cellular processes such as cell cycle progression, cell growth, differentiation, and apoptosis. Transcriptional expression of the σ isoform of 14-3-3 is frequently impaired in human cancers, including carcinomas of the breast, which has led to the suggestion that this protein might be involved in the neoplastic transformation of breast epithelial cells. Here we report on the analysis of 14-3-3σ expression in primary breast tumors using a proteomic approach complemented by immunohistochemical analysis by means of specific antibodies against this isoform. We show that the levels of expression of 14-3-3σ were similar in non-malignant breast epithelial tissue and matched malignant tissue with only sporadic loss of expression observed in 3 of the 68 tumors examined. Moreover we show that 14-3-3σ immunoreactivity was restricted to epithelial cells and significantly stronger in the myoepithelial cells that line the mammary ducts and lobules. The lack of expression of 14-3-3σ in the three breast carcinomas was not associated with high levels of expression of the dominant-negative transcriptional regulator ΔNp63 or with increased expression of estrogen-responsive finger protein, a ubiquitin-protein ligase (E3) that targets 14-3-3σ for proteolysis. Validation of the results was performed retrospectively on an independent clinical tumor sample set using a tissue microarray containing 65 primary tumors. Our data suggest that, contrary to what was previously thought, loss of expression of 14-3-3σ protein is not a frequent event in breast tumorigenesis.