Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with Factin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.Myosin is a molecular motor that interacts with actin filaments and converts chemical energy of ATP to mechanical work. The molecular structure of conventional myosin is characterized by its globular head domain and the filament forming ␣-helical coiled-coil tail domain. Although the former characteristic is shared by a number of unconventional myosins, only conventional myosin, classified as the second class of myosin in myosin super family (1-6), carries the latter one. The amino acid sequence of the tail shows the seven-residue repeat, characteristic of coiled-coil proteins (7), and in addition to this, the myosin tail shows a 28-residue repeat in which charged residues face toward the outside of the coiled-coil structure (8 -11). Because filament formation of myosin is abolished at high ionic strength, these charged residues in the tail have been thought to be responsible for the assembly of myosin molecules. Use of proteolytic fragments (12, 13) as well as recombinant expressed fragments (14 -16) has revealed that the C-terminal region of LMM 1 is important for the assembly of the rod. For skeletal myosin, deletion of the N-terminal residues of LMM did not affect the solubility at low ionic strength, but deletion of 92 residues from the C terminus caused an increase in solubility (16). Hodge et al. (17) reported that, by expressing the rod portion of nonmuscle myosin II by Escherichia coli expression system, the 35-residue C-terminal nonhelical region influences filament formation. Recently it was reported that the deletion of 29 residues near the C terminus of skeletal myosin heavy chain disrupts the filament formation (18). On the other hand, for amoeba myosin, the C-ter...