The 68-residue IA(3) polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic alpha-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K(i) < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA(3) from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA(3). The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained.