A novel matrix metalloproteinase-26 (MMP-26) is known to be specifically expressed in epithelial carcinomas. To facilitate studies of MMP-26 transcriptional regulation, we have cloned and characterized a 1 kb 5'-flanking region of the human MMP-26 gene. Altogether, our findings indicate that the MMP-26 promoter has distinctive structural and functional features among MMP genes. An unusual polyadenylation site proximal to the transcription-factor-binding sites protects transcription of the MMP-26 gene from the upstream promoters and represents a part of the stringent transcriptional regulation of the gene. The MMP-26 gene has a consensus TATA-box and one transcriptional start site located 60 and 35 nucleotides upstream of the translational start site, respectively. The MMP-26 promoter was able to drive luciferase expression in human A549 lung carcinoma, HT1080 fibrosarcoma and HEK293 embryonic kidney cells. The basal transcription efficiency of the MMP-26 promoter is relatively low, thereby explaining the minute expression of the gene in most cells and tissues. When compared with other MMP genes, the MMP-26 promoter contains binding sites for a few transcription factors. Sequential deletion and mutation analysis, and electrophoretic mobility-shift assay have identified the T-cell factor-4 (Tcf-4) motif and the activator protein-1 site as the major regulatory elements of the MMP-26 promoter. Since previous studies have established that the Tcf-4 transcription factor is subjected exclusively to regulation through the beta-catenin/E(epithelial)-cadherin pathway, this implies the specific expression of MMP-26 in cancer cells of epithelial origin.