Agaricus bisporus agglutinin (ABA) is known as a useful lectin to detect T-antigen (Core1) disaccharide (Galbeta1-3GalNAcalpha) and related O-linked glycans. However, a recent X-ray crystallographic study revealed the presence of another intrinsic sugar-binding site, i.e., for GlcNAc. To confirm this possibility, detailed analysis was performed using two advanced methods: lectin microarray and frontal affinity chromatography (FAC). In the lectin microarray, intense signals were observed on ABA spots for both N-glycanase-treated and O-glycanase/beta1-4galactosidase-treated Cy3-labeled asialofetuin. This indicates substantial affinity for both O-linked and agalactosylated (GlcNAc-exposed) N-linked glycans. A further approach by FAC using 20 pNP and 130 PA-oligosaccharides demonstrated that ABA bound to Core1 (K(d) = 3.4 x 10(-6) M) and Core2 (1.9 x 10(-5) M) but not to Core3 and Core6 O-linked glycans. It also showed substantial affinity to mono-, bi-, and tri-antennary agalactosylated complex-type N-linked glycans (K(d) > 1.8 x 10(-5) M). These results establish ABA as a lectin having dual sugar-binding sites with distinct specificity, i.e., for Gal-exposed O-linked glycans and GlcNAc-exposed N-linked glycans.