We have examined whether or not mutations exist in the proximal promoter region of the human alpha-fetoprotein (AFP) gene in the hepatocellular carcinoma (HCC) tissue. Genomic DNA was extracted from four patients: one HCC tissue, one HCC and its corresponding non-cancerous (cirrhosis) tissues, one liver cirrhosis (LC) tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. Serum concentrations of AFP in the patients ranged from less than 5 to 10138 ng/ml. Nucleotide sequence was determined by direct sequencing using a single-stranded DNA template that was produced first through the polymerase chain reaction (PCR) amplification and then asymmetric PCR. In one HCC tissue taken from the patient with a high concentration of serum AFP, nucleotides different from published ones were detected at -120 and -113. These changes, however, probably reflect a DNA polymorphism, because peripheral blood leukocytes of the same patient had the same changes. Including this patient, no mutations in the region from -160 to -10 were detected in the HCC specimens we have examined. These results suggest that the extremely proximal promoter region of the AFP gene where glucocorticoid-responsive element and HNF-1 binding sites exist is not responsible for the re-expression of AFP in HCC.