Specific molecular-receptor interactions with gut epithelium cells are important in understanding bioactivity of food components and drugs, binding of commensal microflora, attachment and initiation of defense mechanisms against pathogenic bacteria and for development of targeted delivery systems to the gut. However, methods for probing such interactions are lacking. Methodology has been developed and validated to measure specific molecular-receptor interactions on living human colorectal cancer cells as in vitro models for the gut epithelium. Atomic force microscopy (AFM) was used to measure ligand–receptor interactions and to map receptor locations on cell surfaces. Measurements were made using silica beads attached to the AFM tip-cantilever assembly, which were functionalized by coupling of ligands to the bead surface. Wheat germ agglutinin (WGA) binds to the glycosylated extracellular domain III of the epidermal growth factor receptor. Methodology was tested by measuring binding of WGA to the surface of confluent monolayers of living Caco-2 human intestinal epithelial cells. The measured modal detachment force of 125 pN is typical of values expected for single molecule interactions. Adhesive events were used to map the location of binding sites on the cell surface revealing heterogeneity in their distribution within and between cells within the monolayer.—Gunning, A. P., Chambers, S., Pin, C., Man, A. L., Morris, V. J., Nicoletti, C. Mapping specific adhesive interactions on living human intestinal epithelial cells with atomic force microscopy.