The pituitary tumor transforming gene-binding factor (PBF) is a poorly characterised gene that is over expressed in pituitary and thyroid tumours. Recently we showed that subcutaneous expression of PBF elicits tumours in nude mice, and expression correlates with thyroid tumour recurrence in man. Given the established role of ionising radiation in thyroid tumourigenesis we have now investigated the relationship between PBF and the tumour suppressor protein p53. Initially, we demonstrated that PBF can influence p53-mediated gene regulation through Hdm2 promoter assays in p53-null H1299 cells. Transfection of p53 elicited a 143±17-fold stimulation of promoter activity. However, co-transfection of PBF significantly repressed p53 transcriptional activity (41±5-fold; P<0.001). Exposure of wild type mouse primary thyrocytes to γ-irradiation resulted in significantly increased PBF protein expression after 24 h (1.88±0.09-fold; P<0.017). Co-immunoprecipitation assays revealed direct binding of PBF and p53 in vivo with a marked increase in binding after treatment with γ-irradiation. Furthermore, through immunofluorescence studies, COS-7 and HCT116 cells treated with γ-irradiation showed nuclear localisation of PBF compared to controls, resulting in an enhanced degree of colocalisation of both PBF and p53 within the nucleus. HCT116 cells transiently overexpressing PBF demonstrated increased p53 stabilisation within the nucleus from 2 h after treatment, with increased stabilisation persisting for 24 h compared to mock-transfected controls. Finally, our murine model of targeted PBF overexpression in the thyroid displayed a significant increase in p53 stabilisation compared to age-matched wild-type mice (3.7±0.9-fold; P=<0.0048). Taken together, these results highlight a potential role for PBF within the DNA damage response, where PBF stabilises in response to genotoxic stress, binds directly to p53 and inhibits p53-mediated responses. From these findings we propose a novel mechanism in thyroid cancer whereby aberrant regulation of PBF leads to the functional inactivation of p53, thus promoting tumourigenesis.