The clinical management of pancreatic disease is often hampered by a lack of tissue diagnosis. Endoscopic pancreatography offers the opportunity to investigate exfoliated cells. However, the significance of mere cytological investigation is compromised by an insufficient sensitivity. The evaluation of the molecular background of carcinogenesis hopefully is capable of providing more sensitive diagnostic markers. The p16INK4a-/retinoblastoma tumour-suppressive pathway has been shown to be involved in the development of near to all pancreatic neoplasms. p14ARF is another tumour suppressor located in the immediate neighbourhood of p16INK4a. Promoter methylation has been demonstrated to be a major inactivating mechanism of both genes. We sought to further evaluate the role of the gene locus INK4a methylation status in the endoscopic differentiation of chronic inflammatory and neoplastic pancreatic disease. Pancreatic fluid specimens of 61 patients with either pancreatic carcinoma (PCA: 39), chronic pancreatitis (CP: 16) or a normal pancreatogram (NAD: 6) were retrieved. In order to detect methylation of either the p14ARF or the p16INK4a promoter a methylation-specific PCR protocol was applied. While 19 out of 39 patients with PCA showed p16 promoter methylation (49%), none of the 16 patients with CP revealed p16 promoter methylation. p14ARF methylation was found in a lower percentage of PCA specimens and in none of the samples of patients with CP. These results suggest a specific significance of INK4a for the development of malignant pancreatic disease. Our data further indicate a potential role for INK4a methylation as a diagnostic marker in the endoscopic differentiation of benign and malignant pancreatic disease.