Updated project metadata. The aim of this study was the identification of biomarkers for the immunohistochemical diagnosis of cholangiocellular carcinoma (CCC). CCC is a primary cancer which arises from the epithelial cells of the bile ducts and is characterised by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analysed by two-dimensional differential in-gel electrophoresis (2D-DIGE) and by mass spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins which were found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p-value ≤ 0.05), 15 candidate proteins were chosen for verification by immunohistochemistry in an independent cohort of 14 patients. This confirmed the significant up-regulation of chloride intracellular channel protein 1 (CLIC1), gelsolin (GSN), pyruvate kinase isozymes M1/M2 (PKM2), serpin H1, 14-3-3 protein sigma (SFN), stress-induced phosphoprotein 1 (STIP1) and tax1-binding protein 3 (Tax1BP3) in tumorous cholangiocytes when compared to normal hepatocytes, whereas fatty acid-binding protein 1 (FABP1) and Betaine-homocysteine S-methyltransferase 1 (BHMT) were significantly down-regulated. Furthermore, STIP1 and SFN were able to differentiate between CCC tumour cells and non-tumorous cholangiocytes with a sensitivity of 100% and 86%, respectively and a specificity of 100% in both cases. We therefore conclude that these proteins should be considered as potential diagnostic biomarkers for CCC in an immunohistochemical application. Additional experiments addressing the validation of these novel biomarker candidates are being performed.