Background - Inactivation of the tumour suppressor gene p16 (CDKN2/MTS-1/INK4A) and K-ras mutations are among the most frequent genetic alterations in human malignancies. Aims - To investigate the tumour suppressor gene p16 and its possible association with K-ras mutations in intrahepatic cholangiocarcinomas of the liver. Methods - The status of p16 was evaluated in 41 cholangiocarcinomas by methylation specific polymerase chain reactions, micro-satellite analysis, DNA sequencing, and immunohistochemical staining. K-ras mutations were determined by direct DNA sequencing analyses after micro-dissection. The results obtained were correlated with histopathological variables and patient survival. Results - Hypermethylation of the 5' CpG island of the p16 gene was found in 34 of 41 (83%) carcinomas. Homozygous deletion at the p16 region was present in two (5%), and loss of heterozygosity (LOH) in eight cases (20%). We failed to detect p16 gene missense mutations. K-ras mutations were found in 22 of 41 (54%) cholangiocarcinomas and in two cases of tumour surrounding non-neoplastic liver tissue. All 22 cancers with K-ras mutations also exhibited methylated p16. We failed to observe a correlation between K-ras or p16 status and histopathological factors or prognosis of patients. Conclusion - These data suggest that inactivation of the p16 gene is a frequent event in cholangiocarcinoma. The most common somatic alteration is promotor methylation of the p16 gene which is closely associated with K-ras mutations. We failed to establish p16 or K-ras status as independent prognostic factors in these tumours.