AbstractNon‐covalent semisynthetic ribonuclease analogues are used to study the role of histidine‐119 in the active site of RNase A. The solid‐phase synthesis of three RNase 111‐124 peptides, in which histidine‐119 is replaced by 3‐(3‐pyrazolyl)‐L‐alanine,Nπ‐methyl‐L‐histidine andNτ‐methyl‐L‐histidine, respectively, is described. The binding of these peptides to RNase 1‐118 protein is examined. The enzymatic activities of the resulting complexes towards first and second step substrates and their binding to the inhibitor 3′‐cytidine monophosphate are determined.TheNπ‐methylhistidine‐ and the 3‐(3‐pyrazolyl)alanine RNase analogues are devoid of catalytic activity. TheNτ‐methylhistidine analogue is found to be enzymatically active, using yeast ribonucleic acid and 2′,3′‐CMP as substrates. With the latter substrate, kinetic parameters at pH 6.0 have been determined. The results provide strong evidence to suggest that thepros‐nitrogen atom of His‐119 plays an essential role as acid/base catalyst in the enzymatic reaction of RNase. There are indications that thetele‐nitrogen atom of histidine‐119 in the natural enzyme – besides effecting the right pKvalue at thepros‐nitrogen centre ‐ may also contribute by favouring the optimal steric orien