The messenger RNA and DNA methylation of the alpha-fetoprotein gene were studied in 101 resected primary hepatocellular carcinomas, of which 93 were unicentric and 8 were multicentric. Fifty-five were 5 cm or less in diameter (small) and 46 were more than 5 cm in diameter (large). In 48.5% of the cases, we detected alpha-fetoprotein messenger RNA in hepatocellular carcinomas, more frequently in large (60.9%) than in small (38.2%; p < 0.00001) but not in any of the nontumorous livers. The alpha-fetoprotein messenger RNA was detected in 83%, 70% and 6.8% of patients with serum alpha-fetoprotein levels of 320 ng/ml or more, 100 to 319 ng/ml and less than 100 ng/ml, respectively. This finding suggests that alpha-fetoprotein gene expression in hepatocellular carcinoma contributes to the serum alpha-fetoprotein elevation in patients with hepatocellular carcinoma. alpha-Fetoprotein messenger RNA appeared as a major band of 2.4 kb, with two minor species of about 6.5 and 3.6 kb in the hepatocellular carcinoma and the fetal liver. Hypomethylation of the 5' end of the alpha-fetoprotein gene was detected in 78.3% of hepatocellular carcinomas expressing alpha-fetoprotein messenger RNA but infrequently (16.7%) in hepatocellular carcinomas with no detectable alpha-fetoprotein messenger RNA (p < 0.0003). This finding suggests that hypomethylation at the 5' region of the gene is associated with alpha-fetoprotein gene reexpression in hepatocellular carcinoma. The alpha-fetoprotein gene expression helped to differentiate unicentric from multicentric hepatocellular carcinomas and to identify other hidden alpha-fetoprotein-secreting hepatocellular carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)