Cathepsin K(EC 3.4.22.38) is a lysosomal cysteine protease that is strongly implicated in bone resorption. The human cathepsin K gene is highly expressed in osteoclasts and gene mutations cause pycnodysostosis, an autosomal recessive skeletal dysplasia. To investigate the evolutionary relatedness of cathepsin K across species, the mouse cathepsin K gene was isolated. A mouse heart cDNA clone, pMCatKl, contained the 3' untranslated region, mature enzyme coding sequence, and most of the propeptide. The remainder of the gene was amplified from mouse melanocyte RNA using 5' rapid amplification of cDNA ends. The gene contained a 990-bp open reading frame, predicting a 329-amino-acid prepropolypeptide. The structure of the protein included a 15-amino-acid presignal, a 99-amino-acid proregion, and a 215-amino-acid mature enzyme. Two potential N-glycosylation sites were identified, one in the proregion and one in the mature enzyme. The 5' untranslated region was 135 bp. The 3' untranslated region was 470 bp including a 9-bp poly(A) tract and contained two polyadenylation signals. The mouse cathepsin K nucleotide and amino acid sequences were highly conserved with the human, rabbit, and chicken homologues across the proregion and mature enzyme. The mouse cathepsin K gene was isolated from an V129 genomic library, and characterization of its genomic structure and intron sizes revealed exons with the initiation ATG in exon 2 and termination TGA in exon 8, a genomic organization that was highly conserved with its human homologue. The availability of the mouse cathepsin K cDNA and genomic sequences will facilitate generation of a mouse model of cathepsin K deficiency by gene targeting.