Association and dissociation kinetics of antibody-hapten interactions of high affinity and specificity have been determined by newly developed techniques using dextran-coated charcoal for rapid separation of free and antibody-bound hapten. Interactions of 12 combinations of four antibody populations (rabbit digoxin-specific antibody, sheep digoxin-specific antibody, rabbit ouabain-specific antibody, and rabbit digitoxin-specific antibody) with three haptens ([3-H]digoxin, [3-H]ouabain, and [3-H]digitoxin) have been studied in terms of both association and dissociation kinetics, and compared in selected instances with association constants determined under equilibrium conditions. Association rate constants determined under both second-order and pseudo-first-order conditions were found to be similar for all antibody-hapten pairs studied (range 0.87-1.7 times 10-7 M-minus 1 sec- minus 1), and were comparable to values previously estimated for antibodies to dye haptens of markedly lower affinity. In contrast, dissociation rate constants varied markedly from 1.9 times 10- minus 4 to 1.7 times 10- minus 2 sec- minus 1. Ratios of association to dissociation rate constants measured by these methods were in satisfactory agreement with average intrinsic association constants measured under equilibrium conditions. These studies support the concept that the major kinetic variable governing antibody-hapten interactions is the rate of dissociation of the complex, and that the strength of antibody-hapten association is determined principally by the activation energy for dissociation.