The aim of this study was to identify genes involved in the development of borderline and malignant phyllodes tumours of the breast (PTs). Expression profiling of 23 PTs (12 benign, 11 borderline/malignant) was performed using Affymetrix U133A GeneChips. mRNA expression in the borderline/malignant PTs was compared to the benign PTs. A group of 162 genes was over-expressed in the borderline/malignant group with a fold change > 2 and FDR < 0.1. Four of these genes were chosen for further investigation:PAX3, SIX1, TGFB2andHMGA2. Over-expression was validated in a separate set of formalin-fixed, paraffin-embedded (FFPE) tumours, using eitherin situhybridization or immunohistochemistry. This confirmed that expression ofPAX3, SIX1, TGFB2andHMGA2in the stromal component of PTs was associated with the borderline/malignant phenotypes (p= 8.7 × 10−5,p= 0.05,p= 0.009,p= 0.003, respectively; Fisher's exact test). The functional consequences of down-regulating these genes were studied using siRNA in short-term cultures and cell lines established from PTs. mRNA ‘knock-down’ ofPAX3resulted in significantly decreased cell proliferation in both a malignant and a borderline PT cell culture. mRNA ‘knock-down’ ofSIX1andHMGA2resulted in decreased cell proliferation only in the malignant PT cell line, and ‘knock-down’ ofTGFB2resulted in decreased cell proliferation only in the borderline PT cell culture. This study shows that these four genes are involved in the development of borderline/malignant PTs.SIX1over-expression was most marked in the highly malignant PTs, with particularly high expression in one case of metastatic PT.PAX3, TGFB2andHMGA2were expressed predominantly in borderline/malignant PTs, but showed some expression in benign tumours; they may be important in the transition from the benign to borderline/malignant phenotype. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.