AbstractCharacterization of polyU immobilized chromatography was performed in order to use this technique as an analytical device. A method of analysis of the elution profile related to thermodynamic parameters was also developed.Sites of attachment of polyU to agarose gel activated by cyanogen bromide were studied using uridine diphosphate and adenine. Independent equilibrium dialysis of ApA for different states of polyU, in solution and immobilized in gel, were carried out. These results show that the immobilized polyU is attached to agarose only at the 5′‐terminal phosphate groups and behaves as it does in solution.Column chromatography of ApA with the immobilized polyU was performed at several temperatures and concentrations. To analyze the elution profile, the theory of cooperative binding of oligonucleotides to polynucleotides was extended to the frame‐work of plate theory. A computer simulation for the elution profiles was performed using thermodynamic parameters obtained by equilibrium dialysis. This simulation duplicated the experimental results. This fact shows that the peculiar leading form of elution profile is due to the cooperative binding. The thermodynamic parameters of the polyU–ApA system were obtained from the “peak tr