Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited lysosomal storage disease involving a mutation in the CLN3 gene. The sequence of CLN3 was determined in 1995; however, the localization of the CLN3 gene product (Cln3p) was not confirmed. In this study, we investigated endogenous Cln3p using two peptide antibodies raised against two distinct epitopes of murine Cln3p. Identification of the liver 60 kDa protein as Cln3p was ascertained by amino acid sequence analysis using tandem mass spectrometry. Liver Cln3p was predominantly localized in the lysosomal membranes, not in endoplasmic reticulum (ER) or Golgi apparatus. As the tissue concentration of brain Cln3p was much lower than that of liver Cln3p, it could be detected only after purification from brain extract using anti-Cln3p IgG Sepharose. The apparent molecular masses of liver Cln3p and brain Cln3p were determined to be about 60 kDa and 55 kDa, respectively. Both brain and liver Cln3p were deglycosylated by PNGase F treatment to form polypeptides with almost the same molecular mass (45 kDa). However, they were not affected by Endo h treatment. In addition, it was also elucidated that the amino terminal region of Cln3p faces the cytosol.