Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths. Deregulation of cell-cycle control is thought to be a crucial event in malignant transformation, and CDC25 phosphatases are a family of cyclin-dependent kinase activators, which act at different points of the cell cycle, including G1-S and G2-M transition. Here, we investigated the expression and functional significance of CDC25s in PDAC. CDC25B mRNA expression levels in human pancreatic tissue samples were analysed by cDNA array, quantitative PCR and Northern blotting. Immunohistochemistry was carried out to localize and quantify CDC25B expression. Two specific CDC25B inhibitors were utilized to determine the functional relevance of CDC25B. By quantitative RT-PCR, CDC25B mRNA was overexpressed in pancreatic cancer (7.5-fold) in comparison to the normal pancreas. Strong nuclear CDC25B immunoreactivity was present in both pancreatic and metastatic cancer samples, and there was a marked increase of the percentage of positive cells in primary cancer (48.6+/-16.3%) and metastatic tissues (71.7+/-3.1%) compared to normal samples (8.3+/-1.8%). Two CDC25B inhibitors reduced the growth of pancreatic cancer cell lines, resulting in the accumulation of phosphorylated CDC2 and G2/M arrest. These findings demonstrate an important role of CDC25B in cell-cycle progression, raising the possibility that inhibition of CDC25B may have therapeutic potential in pancreatic cancer.