We have purified and characterized a novel glycoprotein (r-lamp-3) with an apparent molecular weight (M_r) of 85000 from membranes of triton-filled lysosomes (tritosomes) by the use of immunoaffinity chromatography on a column of monoclonal antibody-Sepharose 4B. r-lamp-3 accounted for approximately 4% of the total proteins in tritosomal membranes. The isoelectric point (pI) of r-lamp-3 was 4.5 and it was shifted to 6.5 after neuraminidase treatment with its molecular weight decreased by about 7000. Pulse-chase experiments in cultured rat hepatocytes using [³⁵S]methionine showed that r-lamp-3 was initially synthesized as a 77000 polypeptide and processed to a mature protein with an M_r of 85000. Upon treatment with endo-β-N-acetylglucosaminidase H (Endo H), the precursor and mature forms were converted to 55000 and 73000 polypeptides, respectively. From the M_r reduction of the precursor form, we estimated the presence of 10-12 N-linked oligosaccharides/r-lamp-3 polypeptide. The data on enzymatic deglycosylation suggested that the mature form of r-lamp-3 contained the same numbers of high mannose-tyep and complex-type N-linked oligosaccharide chains.