Clonal analysis has shown that hepatocellular carcinoma arises from a single cell. However, the clonality of precancerous lesions and adjacent nonneoplastic tissues is not clear. We analyzed a human androgen receptor locus to elucidate the clonal state of liver tissues including post-hepatitic lesions associated with hepatocarcinogenesis. The analysis was based on a restriction fragment length polymorphism involving an androgen receptor locus on the X chromosome, taking advantage of physiologic random inactivation by methylation of 1 of 2 X chromosomes in females during embryogenesis. Clonality was assessed in 79 randomly located tissue samples microdissected from noncirrhotic liver, including a total of 40 morphologically normal sites in 4 normal livers and 39 sites from a single HCV-infected liver. In addition, 51 regenerative nodules, 4 areas of adenomatous hyperplasia, and 18 hepatocellular carcinomas were sampled. All samples were obtained from livers involved by various neoplasms. Eight of forty samples (20.0%) from the four normal livers and 20 of the 39 samples (51.3%) from the single HCV-infected liver showed a monoclonal pattern. Moreover, 30 of 51 regenerative nodules (58.9%) showed a monoclonal pattern. No histologic differences were evident between mono- and polyclonal nodules. On the other hand, the 18 carcinomas and 4 areas of adenomatous hyperplasia all were monoclonal. Mean calculated monoclonal areas of normal liver and liver with chronic hepatitis were 1.1 and 3.3 mm(2). Our results suggest that areas representing a single clone of hepatocytes are present in normal liver, and these progressively expand as changes advance from chronic hepatitis to hepatocellular carcinoma.