The INK4a-ARF (CDKN2A)- locus on chromosome 9p21 encodes for two tumour suppressor proteins, p16INK4a and p14ARF, that act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in tumorigenesis of hepatocellular carcinoma (HCC), we analysed the alterations of p14ARF p16INC4a and p53. After microdissection, DNA of 71 hepatocellular carcinomas was analysed for INK4-ARF inactivation and p53 mutation by DNA sequence analysis, methylation-specific PCR (MSP), restriction-enzyme related polymerase chain reaction (RE-PCR), mRNA expression and immunohistochemistry. In addition, microdeletion of p14ARF and p16INC4a were assessed by differential PCR. Inactivation of p14ARF was found in 11/71 cases (15%), alterations of p16INK4a occurred in 47/71 carcinomas (66%), which correlated with loss of mRNA transcription. Five tumours (7%) had homozygous deletions of the INK4a-ARF locus. We failed to detect specific mutations of both exons. P16INK4a methylation with an unmethylated p14ARF promotor appeared in 39 cases. Mutations of p53 were found in 30 of 71 HCC (42%), and only one of them harboured p14ARF inactivation. We failed to establish alterations of the INK4a-ARF locus or p53 status as independent prognostic factor in these tumours. Our data indicate, that p14ARF methylation occurs independently of p16INK4a alterations in a subset of HCC together with wild type p53. The INK4a-ARF-/p53-pathway was disrupted in 86% of HCC, either by p53 mutations or by INK4a-ARF inactivation, and may have co-operative effects in hepatocarcinogenesis.