Fetal rat liver cells derived from 19-day gestation rats were exposed in culture to the carcinogen, 3'-methyl-4-dimethyl-aminoazobenzene (MDAB) for 3 days and then maintained in medium supplemented with the tumor promoter, phenobarbital (PB). Tumors developed in immunodeficient mice inoculated with cells derived from cultures which had been maintained for more than 8 weeks. Histologically, three types of tumors could be distinguished. One contained epithelial-like cells, which resembled what has previously been described as 'clear' epithelial cells. The second contained cells which were more basophilic, with prominent nuclei and closely resembled the hepatoma cell line Mc-A-R-777. The third group of tumors possessed cells of both varieties. Cell lines derived from these tumors were then characterized by determining their capacity to synthesize and secrete alpha-fetoprotein, albumin and transferrin by measuring the incorporation of 35S-methionine into immunoprecipitates obtained by reaction with the respective specific antibodies and the content of the respective mRNAs were determined by hybridization to cDNAs. The activity of gamma-glutamyl-transpeptidase (GGT) and the liver specific enzyme tyrosine aminotransferase (TAT), as well as the induction of TAT by dexamethasone was also evaluated. The presence of these markers in some of the cell lines strongly suggests that they are derived from parenchymal cells. In contrast, other cell lines which morphologically resemble 'clear' epithelial cells are negative, suggesting that they may be derived from non-parenchymal epithelial cells which exist in the original culture. However, some epithelial-like cell lines derived from tumors of mixed morphology appear different to those established from tumors which contained only epithelial-like cells. These express low levels of transferrin and tyrosine aminotransferase suggesting that they may be more closely related to hepatocytes than those cells which are derived from tumors which originally comprised only epithelial cells. The absence or presence of liver markers correlates with the morphology of the respective cell lines since transferrin and TAT are only present in significant levels in those lines which comprise cells with a morphology resembling hepatoma cell lines. In cell lines which show mixed morphology, immunocytochemistry reveals that significant amounts of transferrin are only present in the parenchymal-like population. Growth rate measurements show that the faster growing cell lines generally possessed lower levels of transferrin and TAT expression. It can be concluded from these studies that it is possible to transform cells derived from fetal rat liver in culture using a hepatocarcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)