Cathepsin L2 is a recently described cysteine proteinase with high sequence homology to cathepsin L and other members of the papain superfamily of cysteine proteinases. Its expression is regulated in a tissue-specific manner and is high in thymus, testis and cornea. In the present study, the entire gene sequence, including 5′ and 3′ flanking region, and chromosomal localization of human cathepsin L2 were determined. The gene spans approximately 6.4 kb and consists of eight exons and seven introns. Genomic organization was similar to human cathepsin Land more than 50% similarity was found between the first introns of cathepsin L and L2, suggesting that they diverged late in evolution. The transcription initiation site, determined by primer extension, was 198 nucleotides from the first ATG. The 5′ flanking region lacks a TATA box but has one SP1 site. The gene was mapped to chromosome 9q21-22 by fluorescence in situ hybridization and the distance from cathepsin Lw as determined to be 15 cM by compiling radiation hybrid mapping results with a genetic map.