Affinity partitioning in aqueous two-phase systems consisting of dextran and dye-liganded polyethylene glycol was employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle (E.C. 1.1.1.27) with Procion Red HE-3B and four structurally related derivatives of this dye in order to follow the significance of the terminal rings of Procion Red HE-3B for the strength of interaction. The study revealed that the arrangement of the two 1-amino-8-naphthol-3,6-disulphonic acid rings seems to be a prerequisite for the interaction of azonaphthol dyes with LDH. The negatively charged sulfonic acid group at the terminal rings of Procion Red HE-3B enhances the affinity of the ligand for LDH significantly. The removal of this sulphonic acid group or splitting off the complete terminal rings decreases the affinity to LDH and improves the competitive effect of NAD+. The results of affinity partitioning are compared with those of affinity chromatography and kinetic data. The usefulness and the choice of parameters of affinity partitioning as an analytical tool to predict the chromatographic behaviour of dye ligands are discussed.