Serum alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC) patients and expression of the protein in cultured HCC cell lines are highly variable. These observations may arise from features correlated with tissue-specific expression of the gene. Extremely strong and potent liver-specific enhancer activity is confined from -4.1 to -3.3 kb Upstream to the human AFP gene in contrast with that of the rodent which exists in three widely separated regions. To understand the tissue-specific expression of AFP, we examined cis-acting elements in the enhancer. Results revealed binding sites for selected liver-enriched transcription factors (LETFs) in both domains A (-4120 to -3756 bp) and B (-3492 to -3300 bp) of the gene. These sites included: one hepatocyte nuclear factor (HNF)-1 and HNF-4, two HNF-3, and two C/EBP binding sites in domain A. An adjacent domain B contained one HNF-3 site and three C/EBP sites Plus a previously identified HNF-1 site. Each of these elements alone has the ability to stimulate heterogeneous promoter activity in a dose-dependent manner when transfected into AFP producing cells. A comparative study showed that the presence of two HNF-1 and one HNF-4 site is a characteristic feature of human but not rodent AFP enhancer. The mRNA levels of the liver-enriched transcription factors (LETFs) were variable in individual HCC cell lines and together with silencer activities may underlie differential expression of the AFP gene. (C) 2004 Elsevier Inc. All rights reserved.