Expression of the gene coding for thymosin beta-4 (Tbeta-4), the major G-actin sequestering peptide in the cell, is regulated mainly at the level of transcription. In this study, we examined the nucleotide sequence of the 5'-flanking region (from -2202 to -881) of the mouse Tbeta-4 gene, and demonstrated that the DNA fragment from -278 to +410 of this gene was capable of directing the expression of a chloramphenicol acetyltransferase reporter gene in NIH3T3 cells. However, expression of the reporter gene in cells cannot be induced by interferon-alpha treatment even though a rapid activation of endogenous Tbeta-4 gene by this cytokine was observed. These results suggest that the projected interferon-stimulated response element (ISRE) might reside in other parts of the mouse Tbeta-4 gene.