In cultured mouse fibroblasts, secretion of the lysosomal cysteine protease, MEP (major excreted protein) is regulated by growth factors and viral transformation. The ability of this protein to be regulated has been attributed to its intrinsic low affinity for the cation-independent mannose 6-phosphate (Man-6-P) receptor (Dong, J., Prence, E. M., and Sahagian, G. G. (1989) J. Biol. Chem. 264, 7377-7383). In this study, the basis for this low affinity was examined. Chromatography on a cation-independent Man-6-P receptor affinity matrix was used to assess relative affinities of Man-6-P-containing oligosaccharides and proteins, and the state of phosphorylation of the oligosaccharides was determined by ion exchange chromatography on QAE-Sephadex. MEP proteins synthesized by normal NIH 3T3 cells or NIH cells transformed with Kirsten sarcoma virus displayed a similar low affinity for the receptor and were found to possess oligosaccharide species with two phosphomonoester moieties. The affinity of these oligosaccharides for the receptor was the same as intact MEP protein and as great as phosphorylated oligosaccharides obtained from lysosomal proteins with the usual high affinity for the receptor. These results indicate that the polypeptide portion of MEP has no effect on binding of the protein to the receptor and that the difference in affinity of MEP and lysosomal proteins with high affinity cannot be attributed to differences in oligosaccharide structure. To investigate this further, we examined the binding characteristics of MEP made by CHO cells. In contrast to mouse MEP, CHO MEP bound to the receptor with high affinity. Partial endoglycosidase H treatment indicated that CHO MEP has two phosphorylated oligosaccharides, whereas the mouse protein has only one. Both oligosaccharides of the CHO cell protein contained two phosphomonoester moieties and displayed an affinity for the receptor that was indistinguishable from that of oligosaccharides of the mouse protein. Conversion of CHO MEP to a one-oligosaccharide species by partial endoglycosidase H treatment produced a protein that displayed low affinity binding similar to that of mouse MEP. A substantial portion of the pool of CHO cell lysosomal protein was also converted to a low affinity ligand by this treatment. Taken together, these results suggest that high affinity binding to the cation-independent receptor involves a divalent interaction with lysosomal proteins that contain two or more phosphorylated oligosaccharides, and that the low affinity of MEP results from an inability to form this multivalent interaction.