We previously reported ((1979) J. Biochem . 85, 747–753) that both of the burst and nonburst heads contain one reactive lysine residue per head, and that only the reactive lysine residue in the burst head is trinitrophenylated in the presence of PP 1 whereas both reactive lysine residues are modified in the absence of PP 1 . In the present study, subfragment one (S-1) prepared from trinitrophenyl (TNP) myosin was subjected to a limited tryptic digestion, a BrCN cleavage, and a thorough tryptic and α-chymotryptic digestion, and the TNP peptides thus obtained were analyzed by SDS-gel electrophoresis and by gel filtration. In the limited digestion, it was found that the reactive lysine residues are both located in the tryptic peptide of 25–27 K daltons containing the N-terminus of the heavy chain and not in the 19–21 K peptide containing reactive thiols. In the BrCN cleavage, it was found that the reactive lysine residues are not located in the 17 K peptide containing the essential arginine residues (Morkin, E., et al. (1979) J. Biol. Chem. 254, 12647–12652). In the thorough digestion, S-1 prepared from myosin modified in the absence of PP 1 gave two equimolar fractions of TNP peptides in Sephadex G-25 column chromatography, whereas that of S-1 prepared from myosin modified in the presence of PP, gave a single fraction of TNP peptide which corresponds in size to one of the two fractions of TNP peptides obtained above. These findings strongly suggest that the chemical structure around the reactive lysine residue in the burst head is different from that in the nonburst head.