An atomic force microscope (AFM) has been used to directly monitor specific interactions between antigens and antibodies. By using AFM probes functionalized with the Fab fragment of an antiferritin antibody, the adhesion forces between the probe and the ferritin-coated substrates were monitored. Fourier analysis of the adhesion forces distribution data suggests quantization of the forces, with a period of approximately 63 pN. The work estimated from this unitary force and its estimated working distance was about half the free energy difference (Delta F-0) relevant to the intrinsic affinity constant (K-0), derived from the kinetic analysis using isothermal titration calorimetry (ITC) of the antibody-antigen interactions. This periodic force was attributed to a single unbinding event between individual antigen and antibody molecules. The probability of adhesive events was strongly influenced by the functionalization mode of the probes. Probes on which vectorially oriented Fab molecules were immobilized, without any spacers, gave much more distinct unbinding force curves than those on which the Fab molecules were randomly fixed.