Guanidination of the nine e-amino groups or picolinimidylation of the α- and e-amino groups yielded active derivatives of DNase. Since modification of the enzyme with these large substituents, which have delocalized positive charges, should have inactivated the protein if amino groups were involved in the catalytic reaction, none of the primary amino groups of DNase can be essential for catalysis. When positive charges on the enzyme were removed by carbamylation of the α-amino group and about seven e-amino groups, the protein had about half of its original activity. But the Ca++ complex of this derivative was almost fully active. Carbamylation of the remaining two amino groups inactivated DNase and Ca++ did not restore activity; therefore, the positive charges on these groups probably help to maintain the active structure of the enzyme. It is not likely that the two amino groups are involved in binding Mn++-DNA, since they do not seem to be freely available for reaction with cyanate. Trinitrophenylation gave results similar to those of carbamylation, but only one amino group of Ca++-free DNase or four to five groups of Ca++-DNase could be trinitrophenylated without inactivation. The neutral, hydrophobic trinitrophenyl groups probably distort the enzyme more than carbamyl groups do. Two to three amino groups resisted trinitrophenylation, which may also indicate that these groups have structural roles. Simplified procedures are described for the chromatographic determination of homocitrulline and for the spectrophotometric titration of free amino groups with 2,4,6-trinitrobenzenesulfonic acid. The α-picolinimidyl, e-guanidino derivative of DNase A was active, and its metal-chelating picolinimidyl group could facilitate the preparation of isomorphous derivatives for x-ray studies.