MUC4 is a transmembrane mucin expressed in pancreatic ductal adenocarcinoma (DAC) in contrast to normal pancreas, and is an independent predictor of poor prognosis in patients with invasive DAC. Our aim was therefore to investigate the mechanisms that control MUC4 expression in pancreatic cancer cells. We focused our study on activator protein (AP)-2alpha transcription factor that acts as a tumour suppressor gene in several cancers. In a series of 18 human DAC, using immunohistochemistry, we confirmed that MUC4 was exclusively expressed in cancerous or preneoplastic lesions in 83% of the samples. On the contrary, AP-2 was mainly expressed by non-tumoural ductal cells (61%) or endocrine cells (67%). Moreover, MUC4 and AP-2 were never found co-expressed suggesting an inhibitory role of AP-2alpha in normal ductal cells. In CAPAN-1 and CAPAN-2 cells, transient AP-2alpha over-expression decreased both MUC4 mRNA and apomucin levels by 20-40% by a mechanism involving inhibition of MUC4 promoter. By chromatin immunoprecipitation and gel-shift assays, we demonstrated that this inhibition involved two AP-2 cis-elements located in the -475/-238 region of the promoter. CAPAN-1 clones, which stably over-expressed AP-2alpha, displayed a strong MUC4 down-regulation (-38 to -100%), a significant decrease of both cell proliferation and invasion concomitant to the up-regulation of p27 cyclin-dependent kinase inhibitor. In conclusion, our data provide evidence that AP-2alpha is an important in vivo negative regulator of MUC4 expression in human pancreatic tissue and that AP-2alpha may play a tumour-suppressive role in pancreatic DAC.