In order to identify a possible enhancer-like regulatory sequence for the human tissue plasminogen activator (tPA) gene, various DNA segments in the 5'-flanking region were ligated to the chloramphenicol acetyltransferase (CAT) reporter gene driven by the tPA gene promoter, and their CAT enhancing activities were measured following transfection to human melanoma-derived Bowes cells that highly express the gene. Major and minor activities were detected in two adjacent upstream sequences, 160 base pairs (bp) (-2288 to -2129) and 102 bp (-2390 to -2289), respectively, and the former was subjected to further analysis. The CAT enhancing activity of the 160-bp sequence was greatly affected by its position and orientation in the constructs and the sequence also functioned weakly with the SV40 promoter. Deletion of any small portion from the sequence abolished the CAT enhancing activity, suggesting that the entire sequence is required for the activity. This sequence did not show a further CAT enhancing activity in Bowes cells treated with the inducers phorbol 12 myristate 13-acetate and dexamethasone and did not function in HeLa or HT1080 cells under any conditions. Taken together, the 160-bp sequence is likely to be responsible for the constitutive and/or cell type-specific expression of the tPA gene in human cells.