Mouse transporter protein (MTP), a small, highly conserved mammalian intracellular membrane protein with four putative transmembrane domains, has been implicated in the transport of nucleosides and/or related molecules across intracellular membranes. The production of recombinant MTP in Saccharomyces cerevisiae alters sensitivity of yeast cells to a heterogeneous group of compounds (e.g., antimetabolites, antibiotics, anthracyclines, ionophores, and steroid hormones) by changing the subcellular compartmentalization of these drugs, suggesting that MTP functions similarly in higher organisms. The present study was undertaken to define the intracellular location of MTP in mammalian cells. Native MTP was not detected by indirect immunofluorescence in cell types that expressed MTP mRNA; therefore, a hemagglutinin (HA) epitope-tagged version of MTP was produced in cultured BHK21 cells by transient transfection, and its distribution within cells was determined by confocal microscopy using antibodies directed against the HA epitope and various organellar proteins. Antibodies directed against HA-MTP colocalized with antibodies against late endosomal and lysosomal proteins but not with antibodies against either Golgi or early endosomal proteins. Analysis of subcellular fractions from rat liver by immunoblotting with antibodies directed against MTP demonstrated the presence of a MTP-like protein in Golgi- and lysosome-enriched membranes but not in mitochondria. These results indicate that MTP resides in late endosomes and lysosomes, a finding that is consistent with the proposed role for MTP in the movement of a variety of small molecules across endosomal and lysosomal membranes. MTP shares a number of characteristics with other lysosome-associated proteins. We, therefore, propose that it be redesignated murine lysosome-associated protein transmembrane 4.