Cathepsin L is a cysteine protease whose overexpression in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic. Regulation of the transcription of the gene encoding human cathepsin L has not been yet studied and only preliminary data exist on the promoter regulation of the gene encoding rodent cathepsin L. In the present study we identified molecular elements involved in the transcriptional regulation of human cathepsin L in melanoma cells. The sequence of the 5′-flanking region of the gene encoding human cathepsin L was determined up to 3263bp upstream of the translation start site. The major transcription intiation site was located. Three mRNA splice variants, differing in their 5′ untranslated ends, were identified. Regulatory regions crucial for cathepsin L promoter activity were characterized between −1489 and −1646bp. In this region, two GC boxes (−1590/−1595 and −1545/−1550) and a CCAAT motif (−1571/−1575) were involved in specific DNA–protein interactions. An electrophoretic mobility-shift assay demonstrated that Sp1 and Sp3 transcription factors bound to these GC boxes, and only the transcription factor nuclear factor Y (NF-Y) bound to the CCAAT motif. Mutagenesis studies demonstrated that these binding sites contributed at least 85% of cathepsin L promoter activity. Thus structural and functional analysis demonstrated that binding sites for NF-Y, Sp1 and Sp3 are essential for transcription of the gene encoding human cathepsin L in melanoma cells.