Recently, we and others identified the 66.3-kDa protein as one of several putative novel lysosomal matrix proteins by analyzing mannose 6-phosphate receptors binding proteins [Kollmann K., Mutenda K.E., Balleininger M., Eckermann E., von Figura K., Schmidt B., Lübke T. (2005) Identification of novel lysosomal matrix proteins by proteome analysis. Proteomics 5(15), 3966-3678, Sleat D.E., Lackland H., Wang Y., Sohar I., Xiao G., Li H., Lobel P. (2005) The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins. Proteomics. 5(6), 1520-1532]. Here, we describe the expression of the mouse 66.3-kDa protein in HT1080 cells in which it is synthesized as a precursor of about 75kDa and subsequently processed by limited proteolysis to mature polypeptides accumulating in the lysosomal compartment. The lysosomal localisation of the endogenous 66.3-kDa protein was verified by indirect immunofluorescence in mouse embryonic fibroblasts and by subcellular fractionation of tyloxapol-filled mouse liver lysosomes. Northern blot analysis reveals high transcriptional levels in testis, liver and kidney, whereas Western blot analysis shows high protein levels in brain, heart, lung and spleen. Interestingly, in mouse the endogenous 66.3-kDa protein is processed in a highly tissue-dependent manner to mature forms.