We previously showed that the -278 to +410 region of mouse thymosin beta4 (mT,beta4) gene supports high levels of reporter gene expression in NIH3T3 cells. This region contains part of the 5'-flanking sequences (-278 to -1), the intact first exon (+1 to +133), and portion of the first intron (+134 to +410). However, the size of this exon is much longer than those of its rat and human counterparts. To resolve the question regarding this size discrepancy, transcription start site for the mTbeta4 gene was re-examined by primer extension and bioinformatics analyses. We found that the first exon of mTbeta4 gene spans 56 bp with its cap site situated in a putative initiator highly similar to the consensus mammalian sequence. In addition, a TATA box-like motif and two consecutive downstream promoter elements were also found. To delineate the cis-elements involved in modulating the constitutive expression of mTbeta4 gene, transient transfection assay was performed. Interestingly, expression level of the reporter gene driven by the -117 to +56 region of mTbeta4 gene was approximately 8-fold higher than that directed by the SV40 promoter and significant promoter activity was found to be associated with the smaller (-56 to +56) fragment. A nuclear protein-bound silencer was located in the region between the -167 and -118 and an enhancer whose effect did not seem to be dependent on protein binding was identified in the downstream (-117 to -88) region. However, neither of these cis-elements affected reporter expression driven by a SV40 promoter. Intriguingly, mTbeta4 promoter functioned well in human colorectal (SW480) and cervical (HeLa) carcinoma cells. Taken together, our findings not only provide crucial information for further elucidation of the transcriptional regulation of mTbeta4 gene but also raise the possibility of utilizing its promoter to produce large quantity of recombinant proteins in mammalian cells.