Alpha-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8-32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration. These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted alpha-lactalbumin. Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of alpha-lactalbumin. Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human alpha-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50. Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives.