Thymosin beta4 (Tbeta4), the actin-sequestering protein that regulates the polymerization and depolymerization of actins, is widely distributed in animal tissues. Expression of this gene in human cells is developmentally regulated and appears to play critical roles in tumorigenesis. As a first step toward analyzing the transcriptional regulation of human Tbeta4 (hTbeta4), we isolated the gene encoding this peptide and characterized its structure features. Human Tbeta4 gene comprises three exons and two introns distributed over 2 kb with its transcription start site at 72 bp upstream of the initiation codon. Expression of the chloramphenicol acetyltransferase (CAT) reporter constructs directed by various parts of the 5'-flanking region of this gene was evaluated by transient transfection assays using human colorectal carcinoma SW480 cells as hosts. Significant promoter activity was found in the -437 to +29 region of hTbeta4 gene even though it lacks both TATA and CCAAT boxes. However, cis-element(s) responsible for PMA-induced expression of Tbeta4 gene was not identified within its 1.5-kb 5'-flanking region. Taken together, our data provide crucial information for further dissection of the molecular mechanism(s) underlying aberrant expression of the Tbeta4 gene during malignant progression of human cancers.