Knowledge of the molecular interactions between lectins and carbohydrates is a key to understand cellular interactions and to develop new bioanalytical applications. We have used atomic force microscopy (AFM) imaging and force measurements to probe the specific interactions between the lectin concanavalin A (Con A) and oligoglucose saccharides. To this end, gold-coated substrates were first functionalized with Con A and thiol-terminated hexasaccharide molecules. The functionalization procedures were validated by means of X-ray photoelectron spectroscopy and AFM. AFM images recorded in aqueous solution revealed that the hexasaccharide-terminated substrates interact specifically with the lectin. Force-distance curves were then recorded between hexasaccharide-terminated AFM probes and Con A-terminated substrates. About half of the retraction curves showed unbinding forces of 96 +/- 55 pN (n = 100), along with elongation forces and rupture lengths ranging from 0 to 200 nm. These features were not observed when the measurements were performed in the presence of mannose or with a hydroxyl-terminated probe. These results, together with the AFM images, indicate that the measured unbinding forces originate from specific lectin-carbohydrate interactions. The carbohydrate AFM probes designed here offer promising prospects for mapping lectin receptors at cell surfaces.