OBJECTIVE Protein Z (PZ), which regulates blood coagulation, is mainly synthesized in the liver. Its plasma level varies widely among individuals, and is highly sensitive to Warfarin. The mechanism for the basic transcription of the human PZ gene, however, has not been reported. The aim of this study was to elucidate the mechanism of gene regulation for PZ by characterizing its 5'-flanking region. RESULTS A reporter gene assay using the human hepatoma cell line, HepG2, identified a minimal promoter region (site A) and two enhancer regions (sites B and C) in the PZ gene. DNase I footprinting and electromobility shift assays revealed binding of the liver-enriched transcriptional factor hepatocyte nuclear factor (HNF)-4alpha to site A, the ubiquitous transcriptional factor Sp1 to sites A and C, and an unidentified factor to site B. The co-transfection of an HNF-4alpha expression vector with reporter gene constructs to the non-hepatic cell line HeLa resulted in a significant increase of PZ promoter activity. CONCLUSIONS HNF-4alpha plays a crucial role in human PZ gene expression in hepatocytic cells, and Sp1 is also important. These findings provide the first step toward understanding the mechanisms of the varying plasma PZ levels in individuals under physiological and pathological conditions.