Aim: Cholangiocarcinoma is generally detected late in the course of disease, and current diagnostic techniques often fail to differentiate benign from malignant disease. Ongoing biomarker studies for early diagnosis of cholangiocarcinoma are still continues. By this study, we analyzed the roles of serum and biliary MMP-9 and TIMP-1 concentrations in the diagnosis of cholangiocarcinoma. Materials and methods: The 113 patients (55 males, 58 females) were included; 33 diagnosed with cholangiocarcinoma (malignant group) and 80 diagnosed with choledocholithiasis (benign group). MMP-9 and TIMP-1 concentrations were analyzed in serum and bile and compared in the malignant and benign groups. Results were evaluated statistically. Results: Biliary MMP-9 concentrations were significantly higher (576 +/- 209 vs. 403 +/- 140 ng/ml, p < 0.01) and biliary TIMP-1 concentrations were significantly lower (22.4 +/- 4.9 vs. 29.4 +/- 6.1 ng/ml, p < 0.01) in the malignant than in the benign group. In contrast, serum MMP-9 and TIMP-1 concentrations were similar in the two groups. Receiver operating curve analysis revealed that the areas under the curve of bile MMP-9 and TIMP-1 were significantly higher than 0.5 (p < 0.001). The sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and accuracy were 0.94, 0.32, 0.36, 0.93, 1.40, 0.19 and 0.5 for biliary MMP-9, respectively, and 0.97, 0.36, 0.39, 0.97, 1.5, 0.08 and 0.54 for biliary TIMP-1, respectively. Conclusion: Serum and biliary MMP-9 and TIMP-1 tests do not appear to be useful in the diagnosis of cholangiocarcinoma.