In hepatocarcinogenesis in rodents, induction of foci and nodules comprising clonally proliferated initiated cells is considered to be essential for the future development of carcinomas. Nodules in human cirrhotic liver, though known to be associated with a high hepatocellular carcinoma risk, have generally been regarded as regenerative in nature, and not the result of clonal or neoplastic cell proliferation, on a morphological basis. However, when we analyzed 83 cirrhotic nodules from 11 HBV carrier patients, utilizing hepatitis B virus (HBV) integration as a marker for clonal proliferation, we found the existence of clonal populations of more than 10(5) hepatocytes in 26 (31.3%) of them. Although such clonal cell populations are morphologically not discernible from neighboring hepatocytes, they may have particular histogenetic significance in human hepatocarcinogenesis and clearly deserve further investigation. Allelotype analysis of mouse hepatocellular carcinomas (HCC), induced by a single dose of diethyl nitrosaminine in C3H/MSM F1 hybrids, revealed no remarkable alterations in the original tumors when microsatellite probes were used, but loss of heterozygosity of chromosome 4 at extremely high frequency (95%) in cultured cell lines derived from these HCC. The shortest common region was about 10 cM distal to the interferon alpha gene, in which the p16 gene is located. The results indicated that loss of gene function, most probably including that of the p16 gene, may be essential for immortalization of cultured hepatocytes but that it may not play any role in initiation or early events in mouse hepatocarcinogenesis in vivo. The mouse HCC used for analysis in this study may be comparable with human HCC at an early stage, for which only very limited genetic alterations have so far been identified.