Interaction of Vicia villosa agglutinin-B4 (VVA-B4) to glycopeptides with O-linked GalNAc residues was investigated by surface plasmon resonance. The affinity was shown to be influenced by the arrangement of O-glycosylation sites on a peptide, PTTTPITTTTK, representing the tandem repeat of MUC2. The association rate constant was relatively high with a particular category of GalNAc-peptides in which more than three amino acid residues were placed between GalNAc-Thr residues. PTT*T*PITT*T*TK (T* indicates GalNAc-Thr) had the highest association rate constant among the glycopeptides tested. The dissociation rate constant was low in the peptides containing consecutive GalNAc residues and PT*TTPIT*T*T*TK was the lowest of the glycopeptides tested. Dissociation constant (K-D), calculated as k(d)/k(a) was the lowest with PTT*T*PITT*T*TK. Therefore, the arrangement but not the quantity of GalNAc residues apparently determines the affinity between VVA-B4 and peptides with attached GalNAc residues. Crown Copyright (C) 2008 Published by Elsevier Inc. All rights reserved.