␣-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis-histidine-50, glutamic acid-96, and histidine-137-were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the threedimensional structure of these variants of ␣-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of ␣-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type ␣-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of ␣-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by ␣-sarcin.