Homologous mammalian proteins were subjected to an exhaustive search for residues that are critical to their structure/function. Error-prone polymerase chain reactions were used to generate random mutations in the genes of bovine pancreatic ribonuclease (RNase A) and human angiogenin, and a genetic selection based on the intrinsic cytotoxicity of ribonucleolytic activity was used to isolate inactive variants. Twenty-three of the 124 residues in RNase A were found to be intolerant to substitution with at least one particular amino acid. Twenty-nine of the 123 residues in angiogenin were likewise intolerant. In both RNase A and angiogenin, only six residues appeared to be wholly intolerant to substitution: two histidine residues involved in general acid/base catalysis and four cysteine residues that form two disulfide bonds. With few exceptions, the remaining critical residues were buried in the hydrophobic core of the proteins. Most of these residues were found to tolerate only conservative substitutions. The importance of a particular residue as revealed by this genetic selection correlated with its sequence conservation, though several non-conserved residues were found to be critical for protein structure/function. Despite voluminous research on RNase A, the importance of many residues identified herein was unknown, and those can now serve as targets for future work. Moreover, a comparison of the critical residues in RNase A and human angiogenin, which share only 35% amino acid sequence identity, provides a unique perspective on the molecular evolution of the RNase A superfamily, as well as an impetus for applying this methodology to other ribonucleases.