Substructure of the myosin rod and its correlation to filament formation is largely based on studies of proteolytic digests and expressed proteins. However, tryptic digestion of myosin always produces polymorphous peptides. Consequently, it is difficult to determine the relation between myosin substructure and filament formation. Similarly, filament formation with recombinant myosin protein is also difficult to interpret because it is never clear whether the recombinant protein folds like the native protein. We recently reported a novel metal protease isolated from squid liver, astacin-like squid metalloprotease (ALSM), which can specifically hydrolyze in vitro myosin heavy chain. In the present study, we examined the solubility properties of the 65-kDa peptide and light meromyosin (LMM) prepared by ALSM isoform II and trypsin digestion, respectively. The 65-kDa peptide is much less soluble than LMM under physiological conditions, even though the length of 65-kDa peptide is shorter than that of LMM. These results suggest that a novel substructure of myosin drives filament assembly.