Overexpression of cathepsin L, a cysteine protease, and consequently procathepsin L secretion switch the phenotype of human melanoma cells to highly tumorigenic and strongly metastatic. This led us to identify the DNA regulatory sequences involved in the regulation of cathepsin L expression in highly metastatic human melanoma cells. The results of the present study demonstrated the presence of regulatory sequences in the 3' region downstream of the cathepsin L gene and in the 3'- and 5'-flanking regions of GC/CCAAT sites of its promoter. In addition, we established that the 5'-UTR (untranslated region) was the most important region for cathepsin L expression. This 5'-UTR integrated an alternative promoter and sequences involved in post-transcriptional regulation. Transfection experiments of bicistronic reporter vectors and RNAs demonstrated that the cathepsin L 5'-UTR contained a functional IRES (internal ribosome entry site). This complete IRES was present only in one of the three splice variants, which differed in their 5'-UTR. Then, we analysed cathepsin L expression in this human melanoma cell line grown under hypoxia. We demonstrated that under moderate hypoxic conditions (1% O2) intracellular expression of cathepsin L was up-regulated. Hypoxia significantly increased only the expression of the transcript which contains the complete IRES, but inhibited promoter activity. These results suggest that the presence of an IRES allowed cathepsin L mRNA translation to be efficient under hypoxic conditions. Altogether, our results indicated that in vivo a tumour hypoxic environment up-regulates cathepsin L expression which promotes tumour progression.