Hepatocellular carcinoma (HCC) is a common malignancy worldwide and highly associated with chronic virus-B or -C infection and cirrhosis. Molecular studies have shown high frequency of loss of heterozygosity (LOH) in some specific chromosome regions, but LOH on chromosome 9 in HCC has not been thoroughly investigated. In our investigation of chromosome 9 with 19 polymerase-chain-reaction (PCR)-based polymorphic microsatellite markers, 30 of 48 HCC tissue samples (63%) had LOH, and a distinct common deletion region and a region of loss were identified. The first region was located at the 9p21 region and the minimal deletion region was located between loci D9S1747 and D9S1748. This is a region of approximately 200 kb which includes the p16 tumor-suppressor gene. A region of loss was located on 9p13 to 9q33. The putative tumor-suppressor gene for nevoid-basal-cell-carcinoma syndrome (NBCCS) at 9q22.3 resides within this region. In addition to LOH, 4 HCC cases showed possible homozygous deletions at 9p21 with markers D9S1748, D9S1752 and D9S171 by multiplex PCR analysis. In 3 cases, the minimal region of possible homozygous deletion was approximately 300 kb and was defined between markers D9S1747 and D9S1752. Since this deletion region includes both the p15 and the p16 tumor-suppressor genes, these genes were possibly inactivated by homozygous deletion in HCC. In addition, a second region of possible homozygous deletion was present on the centromeric side of 9p21. However, these changes are not associated with age, gender, size or tumor-cell differentiation. Our data also suggest that inactivation of the p16 and the p15 genes and the possibility of other unknown tumor-suppressor genes located on these defined deleted regions of chromosome 9 may be involved in the pathogenesis of HCC.