AbstractSecreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as α‐sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of α‐sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild‐type α‐sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of α‐sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of α‐sarcin on ribosomes.