K-ras mutations at codon 12 (KRM) have been detected in ∼80% of samples of pure pancreatic juice (PPJ) from patients with pancreatic cancer (PCa) and are a promising potential tumor marker. However, the frequent presence of KRM was reported in PPJ from noncancerous patients as determined by a highly sensitive method, raising questions as to the cancer specificity of this marker. Therefore we evaluated whether the hybridization protection assay (HPA), which can quantitatively determine KRM in PPJ, is useful for the diagnosis of PCa, differentiating from chronic pancreatitis (CP). PPJ was collected endoscopically from 29 patients with PCa, 26 patients with CP, and the 11 cases as the control group. Polymerase chain reaction (PCR) and HPA using an acridinium ester-labeled DNA probe for KRM were performed with DNA extracted from these samples. The results were compared with those obtained by PCR-restriction fragment length polymorphism (RFLP). The mean + 2 SD of chemiluminescence in the control group was 11,020 RLUs. When 11,020 RLUs was taken as the cut-off value, KRM was detected by PCR-HPA in 19 (66%) of 29 of PCa and one (4%) of 26 of CP cases. Analysis of PPJ by PCR-RFLP demonstrated KRM in 22 (79%) of 28 of PCa and five (19%) of 26 of CP cases. However, four of five patients with CP who were KRM-positive by PCR-RFLP were defined as negative by PCR-HPA, suggesting that PCR-HPA is superior to PCR-RFLP for the discrimination between PCa and CP. These findings indicate that quantitative analysis of KRM in PPJ using the PCR-HPA method is a promising approach for the diagnosis of PCa, differentiating from CP with a suitable cut-off value, as in the case with the use of conventional serum tumor marker.