The A chain of ricin is a cytotoxic RNA N-glycosidase that inactivates eukaryotic ribosomes. The contribution of the amphiphilic helix D, which is distant from the active site, to the catalysis of the depurination of the adenosine at position 4324 in 28S rRNA has been examined by systematic deletion of amino acids. Two sets of consecutive two- or three-amino acid deletions of the 12 residues in helix D, a total of 20 mutants, were constructed. All 12 of the amino acids could be deleted in one mutant or another without loss of activity; however, mutations that disrupted the amphiphilicity of the helix led to inactivation of the enzyme. Thus, the minimum contribution of helix D to the structure of the ricin A chain is to provide hydrophobic and hydrophilic surfaces to shield helix E, which has the active-site residues; moreover, no amino acid side chain in helix D makes a specific contribution to the recognition of the RNA substrate or to catalysis; and, finally, phasing of the amino acid deletions can be important to the phenotype of mutants.