Extracellular fungal RNases, including ribotoxins such as alpha-sarcin, constitute a family of structurally related proteins represented by RNase T1. The tautomeric preferences of the alpha-sarcin imidazole side chains have been determined by nuclear magnetic resonance and electrostatic calculations. Histidine residues at the active site, H50 and H137, adopt the Ndelta tautomer, which is less common in short peptides, as has been found for RNase T1. Comparison with tautomers predicted from crystal structures of other ribonucleases suggests that two active site histidine residues with the Ndelta tautomer are a conserved feature of microbial ribonucleases and that this is related to their ribonucleolytic function.