The preparation of immobilized bovine pancreatic ribonuclease by covalent attachment to Sepharose 4B, with and without a spacer arm, is described. The coupling reaction was carried out at two different pH values, 8.5 and 10.5, and the different kinetic properties shown by the resulting preparations probably reflect the influence of the particular amino acid side-chains involved in the covalent coupling of the enzyme to the insoluble matrix. The strength of binding of mononucleotides, at 4°C, as deduced from the salt concentration at which they are eluted from an immobilized RNAase column, follows the order 5′-GMP > 5′-AMP > 3′-UMP > 3′-CMP. When binary mixtures of a 3′-pyrimidine nucleotide and a 5′-purine nucleotide are chromatographed jointly, a co-operative effect is found and the elution of either or both ligands is retarded. This behaviour can be explained in terms of the preferential binding of each kind of nucleotide to different sub-sites in the enzyme. The stoichiometry and association constant for 3′-CMP and 5′-AMP at pH 7.0 were also determined. © 1983.