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The chemical shift of the C-2 imidazole protons of the histidine residues of pancreatic ribo-nuclease has been studied as a function of pH in 2H2O. Upon protonation of the imidazole ring the C-2 proton magnetic resonance signal shifts about 1.0 ppm to a lower field. Thus titration curves and approximate pK values of the histidine residues are obtained. The pK values of histidine 119 and histidine 12 of the active site are unusually low and shift by 1.0 and 0.7 units to higher values when the NaCl concentration is changed from 0.1 M to 0.4 M, whereas the pK value of histidine 105 shifts only by about 0.2 units. This indicates the presence of a positively charged group in the immediate environment of histidine 119 and histidine 12. The abnormal chemical shift of the histidine 48 C-2 proton at low pH is explained by means of an interaction of histidine 48 with the carboxylate anion of aspartic acid 14. From an analysis of the titration curves of histidine 119 and histidine 12 the existence of an enzyme species in which both imidazoles arc connected by a hydrogen bond is derived. The C-2 proton magnetic resonance signals of histidine 119 and histidine 12 shift to a lower field when inhibitors bind to the enzyme. Both histidines are then kept in a protonated form up to the pH range of 7–8. The apparent pK values are higher in the cytidine 2′-phosphate-ribonuclease complex than in the cytidine 3′-phosphate- ribonuclease complex. A similar shift of the histidine 119 and histidine 12 signals is observed with binding of pyrophosphate to ribonuclease indicating that only the phosphate group causes the behaviour of the histidines. Shifts of the C-6 proton magnetic resonance signal of the pyri- midine ring indicate an additional binding of the base to an aromatic side chain of the enzyme. The mechanism for the binding process proposed in previous papers by Witzel and coworkers in which histidine 119 and lysine 41 are the binding groups of the enzyme, whereas histidine 12 takes up a proton when being released from its binding to histidine 119, is in agreement with the results reported in this communication. [ABSTRACT FROM AUTHOR]