Nonmuscle myosin II forms a folded conformation (10S form) in the inactivated state; however, the physiological importance of the 10S form is still unclear. To investigate the role of 10S form, we generated a chimeric mutant of nonmuscle myosin IIB (IIB-SK1·2), in which S1462-R1490 and L1551-E1577 were replaced with the corresponding portions of skeletal muscle myosin heavy chain. The IIB-SK1·2 mutant did not fold into a 10S form under physiological condition in vitro. IIB-SK1·2 was less dynamic by stabilizing the filamentous form and accumulated in the posterior region of migrating cells. IIB-SK1·2 functioned properly in cytokinesis but altered migratory properties; the rate and directional persistence were increased by IIB-SK1·2 expression. Surprisingly, endogenous nonmuscle myosin IIA was excluded from the posterior region of migrating cells expressing IIB-SK1·2, which may underlie the change of the cellular migratory properties. These results suggest that the 10S form is necessary for maintaining nonmuscle myosin II in an unassembled state and for recruitment of nonmuscle myosin II to a specific region of the cell.