The carboxypeptidase inhibitor from Russet Burbank potatoes was subjected to a variety of chemical modifications and their effects on inhibitory activity toward carboxypeptidases A and B were determined. The importance of the alpha carboxylate of glycine-39 to the enzyme-inhibitor interaction was demonstrated by the observation that a derivative in which all four carboxyls were modified was inactive whereas a derivative in which only the beta carboxylates of aspartic acid residues 5, 16, and 17 were masked retained full inhibitory activity. In addition to these three aspartic acid residues, lysine residues 10 and 13, histidine residues 3 and 15, and arginine-32 were modified and residues 1-5 removed with little effect on inhibitory activity. Tryptophan residues 22 and 28 did not react with 2-hydroxy-5-nitrobenzyl bromide or o-nitrophenylsulfenyl chloride, and thus are presumed to be buried in the interior of the inhibitor molecule. Although tyrosine-37 was acetylated without affecting binding characteristics, both carboxypeptidases A and B protected against deacetylation by hydroxylamine. These studies indicate that the carboxyl terminal region of the inhibitor is in contact with enzyme in the complex. The parallel effects of modifications on inhibitory activity toward carboxypeptidases A and B support previous evidence that both enzymes utilize the same binding site on the inhibitor [C. A. Ryan (1971), Biochem. Biophys. Res. Commun. 44, 1265].