The INK4a–ARF locus, located on chromosome 9p21, encodes two cell-cycle regulatory proteins, p16INK4a and p14ARF, acting through the Rb–CDK4 and p53 pathways. To study the contribution of each pathway in the tumourigenesis of cholangiocarcinoma, the alterations of p14ARF, p16INK4a, p53, and pRb were analysed. After microdissection, DNAs from 51 cholangiocarcinomas were analysed by methylation-specific PCR (MSP), restriction-enzyme related polymerase chain reaction (RE-PCR), microsatellite analysis, mRNA expression, and DNA sequencing. Immunohistochemistry of p14ARF, p16INK4a, p53, and pRb was also performed. Promoter methylation of p14ARF was found in 13/51 cases (25%) and p16INK4a showed aberrant promoter methylation in 39/51 cases (76%) which correlated with loss of mRNA transcription. Two tumours (4%) had homozygous deletion of the INK4a–ARF locus. Specific mutations of both exons were not detected. p14ARF inactivation appeared in the context of an unmethylated p16INK4a promoter in eight of 13 cases (61%) of the carcinomas methylated at p14ARF. Mutations of p53 were found in 19 of 51 tumours (37%), and four of them (21%) harboured p14ARF inactivation. The pRb protein was detected in 30/51 (59%) tumours examined. The absence of pRB protein did not correlate with any of the examined parameters. Alterations of the INK4a–ARF locus, pRB or p53 status could not be established as independent prognostic factors in these tumours. These findings indicate that the INK4a–ARF locus is frequently inactivated in cholangiocarcinoma of the liver and occurs independently of the status of p53 or pRb. Copyright © 2002 John Wiley & Sons, Ltd.