The details of the interaction between two mutants of Cyanovirin-N (CV-N), an HIV inactivating protein, and di- and trimannosides, substructures of Man-9, were investigated by STD NMR. spectroscopy. One mutant, CV-N-mutDB, contains only one carbohydrate-binding site on domain A, whereas in CV-N-mutDA, the specificity of domain A for trimannose was changed while the site in domain B was kept intact, allowing for a dissection of the overall binding. Results of the STD NMR experiments revealed close contact between the protein binding site on domain A and H2, H3, and H4 of the nonreducing terminal mannose unit for Man alpha(1-2)Man alpha OMe, Man alpha(1-2)Man alpha(1-3)Man alpha OMe, and Man alpha(1-2)Man alpha(1-6)Man alpha OMe. The Man alpha(1-2)Man alpha(1-2)Man alpha OMe trisaccharide interacted with CV-N with the highest affinity. Further dissection of the interaction was achieved by NMR experiments with synthetic 2'-, 3'-, 4'-, and 6'-deoxy analogues of the disaccharide Man alpha(1-2)Man alpha OMe. STD and H-1-N-15 HSQC NMR spectroscopy revealed that the 2'- and 6'-deoxy dimannosides were recognized by CV-N, whereas no binding was detected for the 3'- and 4'-deoxy sugars. These results demonstrate that the 3'- and 4'-hydroxyl groups on the terminal residue are engaged in key polar interactions with the protein and are required for high-affinity binding.