Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor-induced clotting. The TFPI gene contains three GATA motifs in the region flanking its transcription initiation sites. GATA motifs present in promoters of other genes bind GATA-2 transcription factor and thereby regulate their transcriptional expression. Both TFPI and GATA-2 transcription factor are synthesized by a variety of normal as well as malignant cells including hepatocellular carcinoma HepG2 and bladder carcinoma ECV304. Here, we studied whether the three GATA motifs flanking the transcription initiation sites regulate TFPI gene expression in HepG2 and ECV304 cells by binding to the GATA-2 transcription factor. Synthetic oligonucleotides containing GATA sequences from the TFPI regulatory region formed DNA-protein complexes with HepG2 and ECV304 nuclear extracts in an electrophoretic mobility shift assay. Using a 740-bp fragment (-496/+244) from TFPI regulatory region, the effect of base substitutions at each of the three GATA motifs was studied in a luciferase reporter gene system. TFPI promoter activity in HepG2 cells was increased 3-fold with mutation in one of the three GATA motifs and in ECV304 cells was essentially unchanged with mutations in all three GATA motifs. Thus, GATA motifs appear to serve a tissue-specific regulatory role in TFPI gene expression in malignant cells.