The titration curves of the C-2 histidine protons of RNase A and of derivative II-a covalent derivative obtained by reaction of the enzyme with the halogenated nucleotide 9-β-D-ribofuranosyl-6-chloropurine 5'-phosphate-in the presence of a number of purine nucleosides, nucleoside monophosphates, and nucleoside diphosphates were studied by means of proton nuclear magnetic resonance at 270 MHz. The examination of the perturbations found on the chemical shifts and pKs of the C-2 protons of His-12, -48, and -119 are consistent with the following conclusions: (1) The interaction of adenosine in the primary purine binding site of the enzyme (B2R2) induces a conformational change in the active center of the enzyme [for the nomenclature of the RNase A binding subsites, see Parés et al. [Parés, X., Llorens, R., Arús, C., & Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579]]. (2) The phosphate moiety of the ligands, independently of its position, probably acts as a general carrier of the nucleotide to the active center, while the substituents of the base are the generators of the specificity of the binding and control the binding equilibrium between subsites B2R2 and B1R1. (3) There is no overlapping between the binding sites occupied by the labeling nucleotide in derivative II (B3R3p2) and the primary binding site for purine mononucleotides (B2R2P1). © 1982, American Chemical Society. All rights reserved.