Transforming growth factor alpha (TGF-alpha) is expressed at high levels in human hepatocellular carcinoma (HCC); it may be part of a mechanism by which hepatitis B virus (HBV) causes HCC. Research has continued in the analysis of the role of TGF-alpha in the etiology of HCC. Previous work in this laboratory had shown high levels of TGF-alpha expression in most human HCCs, and an up-regulation of the TGF-alpha gene in a hepatoblastoma cell line after transfection with the hepatitis B virus (HBV). Hepatocellular carcinomas (HCCs) often consist of two or more different histologic grades in separate well-defined areas of the same tumor from the same patient. This theoretically provides a model in which to study tissues from the different stages of HCC development. HCCs and adjacent nontumorous livers from 25 patients, including 15 whose tumors contained at least two different histologic grades, were evaluated for the expression of transforming growth factor-alpha (TGF-alpha) and its receptor, epidermal growth factor receptor (EGFR), using an avidin- biotin-peroxidase-complex method. Surgical resections were performed at Kurume University Hospital between 1981-1992. HBsAg was detected in liver in 3/25 (12%). TGF-alpha was detected in 24 of 25 (96%) HCCs and 23 of 24 (96%) available adjacent nontumorous livers. EGFR was detected in 16 of 25 (64%) HCCs and 17 of 24 (71%) adjacent nontumorous livers. Fifteen of the 25 HCCs contained tumor nodules of two different grades (two of the 15 contained nodules of three different grades) which had a less-differentiated histologic grade, arising within or adjacent to the first tumor. In those cases, staining in the less-differentiated area of tumor was usually less intense than in the more highly differentiated area (80% of cases for TGF-alpha; 91% for EGFR). TGF-alpha and EGFR were not detected in two normal livers. These data confirm that increased expression of TGF-alpha and EGFR occur frequently in human HCC. Furthermore, the detection of greater staining in more highly differentiated portions of the tumors suggests that increased expression of TGF-alpha and EGFR may be events of the early stages of human hepatocarcinogenesis.