Myosin II molecules assemble into filaments through their C-terminal rod region, and are responsible for several cellular motile activities. Three isoforms of nonmuscle myosin II (IIA, IIB and IIC) are expressed in mammalian cells. However, little is known regarding the isoform composition in filaments. To obtain new insight into the assembly properties of myosin II isoforms, especially regarding the isoform composition in filaments, we performed a combination analysis of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which enables us to acquire information on both the interaction and the size of each molecule simultaneously. Using C-terminal rod fragments of IIA and IIB (ARF296 and BRF305) labelled with different fluorescent probes, we demonstrated that hetero-assemblies were formed from a mixture of ARF296 and BRF305, and that dynamic exchange of rod fragments occurred between preformed homo-assemblies of each isoform in an isoform-independent manner. We also showed that Mts1 (S100A4) specifically stripped ARF296 away from the hetero-assemblies, and consequently, homo-assemblies of BRF305 were formed. These results suggest that IIA and IIB can form hetero-filaments in an isoform-independent manner, and that a factor like Mts1 can remove one isoform from the hetero-filament, resulting in a formation of homo-filaments consisting of another isoform.