Membrane-type 1 matrix metalloproteinase (MT1-MMP) expressed in tumor cells is believed to be important for the pericellular degradation of extracellular matrices during invasion and metastasis. To analyze the mechanism by which MT1-MMP becomes expressed in cancer cells, we assessed the MT1-MMP promoter region for the presence of cis-acting promoter elements that support transcription in transformed cells. Our tumor model consisted of Madin-Darby canine kidney (MDCK) cells transformed by v-src (src4 cells). MT1-MMP mRNA was only faintly detected in parental cells but was strongly expressed in the src4 cells. In parallel, src4 cells invaded into collagen gels, whereas MDCK cells did not. When MDCK and src4 cells were transiently transfected with a plasmid containing of -3000 to -99 nt from the upstream region of the MT1-MMP gene, the promoter activity was 2.6-fold higher in src4 cells than in MDCK cells. Furthermore, the region between -399 and -356 nt was found to contain the src4-specific enhancer element(s). Tandem Sp1 binding sites were also found to be essential in promoting transcription. An Egr-1 site that partially overlaps with the Sp1 sites was found to cooperate with the src4-specific enhancer and to also contribute weakly to the basal promoter activity. The presence of transcription factors that bind to the src4-specific enhancer site was detected by mobility-shift assays in src4 cell nuclear extracts but only weakly in MDCK extracts. Thus, we have identified a novel enhancer element that acts specifically in the transformed cells to enhance MT1-MMP expression.