Modification of human placental aldose reductase by iodoacetate (IAA) led to a mol/mol binding of IAA, a 40% decrease in the kcat, a 3-5-fold increase in the Km,NADPH and Km,glyceraldehyde and a 600-fold increase in the Ki,sorbinil; determined at pH 6.0. NADPH and 2'-monophosphoadenosine 5'-diphosphoribose but neither glyceraldehyde nor sorbinil, prevented carboxymethylation-induced changes. Cleavage of [14C]IAA-modified enzyme by trypsin resulted in two radiolabeled peptides: Val-297 to Lys-307 and Val-297 to Phe-315. In both these peptides Cys-298 was the only radiolabeled residue. It is suggested that Cys-298 regulates the kinetic and inhibition properties of the enzyme, but does not participate in catalysis.