Kinetic studies of the binding of Co-57-labeled cyanocobalamin to purified chicken serum haptocorrin resulted in an apparently diffusion-controlled second-order rate constant, k(b), of 2.36 x 10(8) M(-1) s(-1) at 25.0 degrees C, pH independent in the range 7.4-9.6, and Delta S double dagger(b) = -4.48 +/- 0.34 cal mol(-1) K-1 and Delta H double dagger(b) = 4.71 +/- 0.10 kcal mol(-1) (5-58 degrees C). Cyanide trapping of the slowly released cyanocobalamin at 85.0 degrees C and pH 7.4 resulted in a concentration-independent rate constant, k(d), of (5.58 +/- 0.17) x 10(-5) s(-1) for dissociation of the cyanocobalamin-haptocorrin complex, with Delta S double dagger(d) = 13.4 +/- 1.6 cal mol(-1) K-1 and Delta H double dagger(d) = 32.9 +/- 0.6 kcal mol(-1) (70-95 degrees C). This lends to a binding constant, K-b, of 5 x 10(16) M(-1) or log K-b = 16.7 +/- 0.6 at 25.0 degrees C, which is the largest protein-ligand binding constant yet reported and at least an order of magnitude higher than that for the biotin-avidin system.