Various alcohol and lactate dehydrogenases were adsorbed to an affinity column of immobilized N6-(6-aminohexyl)-AMP and subsequently eluted with gradients of coenzymes or coenzyme fragments. A linear relation was observed between the eluting concentration of nucleotide and the reported dissociation constants for the corresponding binary enzyme-nucleotide complexes. This relation has been utilized to determine unknown dissociation constants by affinity chromatography. The method presented can also be utilized for the estimation of dissociation constants betweendehydrogenases and coenzyme analogues.