Ninhydrin (1)2,3-indantrione monohydrate) was shown to react with the guanidino group of Nalpha-benzyloxycarbonylarginine under mild conditions (pH 8.0, 25 degrees). When ribonuclease A [EC 3.1.4.22] was reacted with ninhydrin under similar conditions, rapid inactivation took place with concomitant modification of arginine and lysine residues. Specific modification of arginine residues in the enzyme could be achieved by reversible blocking of amino groups with citraconic anhydride. Ribonuclease T1 [EC 3.1.4.8] was also inactivated rapidly by ninhydrin under similar conditions. In this case, the single arginine residue (Arg-77) and the amino groups of the N-terminal alanine and lysine-41 appeared to be specifically modified. Other amino acid residues did not appear to be significantly modified by ninhydrin in either of these enzymes. Ninhydrin thus can be used for the specific modification of arginine residues in proteins under mild conditions by reversibly blocking amino and, possibly, thiol groups.