Interactions of Surfactant Protein D (SP-D) with microorganisms and organic antigens involve binding to the trimeric, neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each with identical N-terminal tags remote from the ligand binding surface. Although rat and mouse showed similar affinities for saccharide competitors, both differed markedly from the human protein. The human neck+CRD preferentially recognized N-acetyl-mannosamine, while the rat and mouse proteins showed greater affinity for myo-inositol, maltose, and glucose. Although human neck+CRDs bound to maltosyl-agarose and fungal mannan, only rat and mouse neck+CRDs showed significant binding to maltosyl-Toyopearl beads, solid-phase maltosyl-albumin neo-glycoprotein, or the Phil82 strain of influenza A virus. Likewise, human SP-D dodecamers and trimeric subunits of full-length rat, but not full-length human SP-D trimers, bound to maltosyl-Toyopearl. Site-directed mutagenesis of the human neck+CRD demonstrated an important role of Asp324-Asp325 in the recognition of N-acetyl-mannosamine, and substitution of the corresponding murine sequence (Asn324-Asn325) conferred the capacity to interact with immobilized maltose. Thus, ligand recognition by human SP-D involves a complex interplay between saccharide presentation, the valency of trimeric subunits, and species-specific residues that flank the primary carbohydrate binding site.