The complete primary structure of the major component myoglobin from the humpback whale, Megaptera novaeangliae, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the acetimidated apomyoglobin at the three arginine residues with trypsin. The further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease enabled the determination of the remainder of the covalent structure. This myoglobin differs from that of sperm whale, Physeter catodon, at 12 positions, and dwarf sperm whale, Kogia simus, at 14 positions, finback whale Balaenoptera physalus at 3 positions, minke whale, Balaenoptera acutorostrata at 2 positions, and California gray whal Eschrichtius gibbosus, at 1 position. All of the substitutions observed in this sequence fit readily into the three-dimensional structure of sperm whale myoglobin.