The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that has been implicated in the induction of differentiation of various cell types, including human uroepithelial cells. PPARgamma-mediated differentiation of normal human urothelial (NHU) cells in vitro requires coinhibition of epidermal growth factor receptor (EGFR) signalling and is characterised by de novo expression of late/terminal differentiation-associated genes, including uroplakins (UPK), over a 6-day period. We used gene microarrays to identify intermediary transcription factors induced in direct response to PPARgamma activation of EGFR-inhibited NHU cells. FOXA1 and IRF-1 contained consensus cognate binding sites in UPK1a, UPK2, and UPK3a promoters and transcripts were induced within 12 h of PPARgamma activation; transcription complex formation was confirmed by electromobility shift assays. In urothelium in situ, both FOXA1 and IRF-1 were nuclear and expressed in a differentiation-associated pattern. Knockdown by transient siRNA of either FOXA1 or IRF-1 abrogated PPARgamma-induced uroplakin expression in vitro. This is the first evidence that ligand activation of PPARgamma induces expression of intermediary transcription factors that mediate an epithelial differentiation programme and represents a new paradigm for understanding differentiation, regenerative repair and inflammation in epithelial tissues.