Mouse P388D1 macrophages target newly synthesized acid hydrolases to lysosomes in spite of their lack of the 215-kDa mannose 6-phosphate (Man-6-P) receptor. We now report that these cells contain a membrane-associated Man-6-P receptor that is distinct from the previously described receptor. The new receptor binds lysosomal enzymes containing phosphomannosyl residues. This binding is inhibited by Man-6-P or by pretreatment of the lysosomal enzymes with alkaline phosphatase. Lysosomal enzyme binding occurs at neutral pH and dissociation of the bound ligand occurs at low pH values comparable to those found within endosomes or lysosomes. The new receptor differs from the 215-kDa Man-6-P receptor in two ways. It has an absolute requirement for divalent cations and is unable to bind Dictyostelium discoideum lysosomal enzymes, which contain methylphosphomannosyl residues rather than the usual phosphomannosyl monoesters. Based on the difference in cation requirement, we suggest that the 215-kDa receptor be referred to as Man-6-P receptor CI (cation independent) and the new receptor as Man-6-P receptor CD (cation dependent). We conclude that the Man-6-P receptor CD functions in the targeting of newly synthesized acid hydrolases to lysosomes in P388D1 macrophages.