The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K(d) = 0.3 +/- 0.1 nm) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d) approximately 500 nm) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.