We examined loss of heterozygosity at 13 loci on 5 chromosomes in hepatocellular carcinomas (HCCs) from 56 patients. In 42 of these cases, regenerative nodules of liver cirrhosis were also analyzed. High frequencies of allelic losses were detected on chromosomes 13q (47%), 16q (40%) and 17p (64%), whereas losses on chromosome 4p and 11p were observed in less than 22% of cases in HCCs. In contrast, LOH was not detected on any loci in cirrhotic nodules. On chromosome 13q, the common region of allelic loss was mapped to the region including the retinoblastoma (RB) locus, by using 8 polymorphic probes. Furthermore, one case with 13q loss had an interstitial deletion of the RB gene, indicating the involvement of inactivation of the RB gene in hepatotumorigenesis. Losses were associated with portal-vein thrombosis or intrahepatic metastasis, increased tumor size, a poorly differentiated phenotype and clinical stage. Losses occurring together on 13q, 16q and 17p were significantly higher in patients in clinical stage IV or histologically poorly differentiated tumors, suggesting that the accumulation of allelic loss occurs in advanced tumors and that patients with multiple allelic losses may have a worse prognosis than those with a single loss.