Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.