The tumor suppressor gene CDKN2A (MTS1/p16), located on chromosome 9p21, is inactivated in a variety of tumors including melanomas and tumors of the biliary tract, pancreas, and stomach. The aim of the present study was to determine whether this gene is inactivated in hepatocellular carcinoma (HCC). Twenty–three primary HCCs and four HCC cell lines were examined. Loss of heterozygosity (LOH) analysis was performed using eight polymorphic markers immediately surrounding CDKN2A, and showed a contiguous region of loss, with the two most commonly deleted markers being D9S1604, located between the p16 and p15 genes, at which 7 of 13 informative tumors (54%) showed loss, and D9S171, with 4 of 14 LOH (29%). Exons 1, 2, and 3 of CDKN2A were amplified by polymerase chain reaction to detect homozygous deletions, and single–strand conformation polymorphism (SSCP) analysis was performed to screen for mutations. No homozygous deletions were detected in any sample. SSCP and sequence analysis showed the same nucleotide change at codon 148 in four tumors. This has been reported elsewhere as a polymorphism. One of these four tumors also contained a mutation at codon 119, resulting in the substitution of an acidic amino acid for a basic one. It is concluded that CDKN2A is infrequently deleted or mutated in HCC. The region of allelic loss upstream from CDKN2A might result in inactivation of regulatory sequences important in the expression of this gene; alternatively, a second tumor suppressor gene may be present in the region 9p21–22, proximal to CDKN2A. These possibilities require further investigation.