Myosin plays a fundamental role in muscle contraction. Approximately 300 myosins form a bipolar thick filament, in which myosin is continuously replaced by protein turnover. However, it is unclear how rapidly this process occurs and whether the myosin exchange rate differs depending on the region of the thick filament. To answer this question, we first measured myosin release and insertion rates over a short period and monitored myotubes expressing a photoconvertible fluorescence protein-tagged myosin, which enabled us to monitor myosin release and insertion simultaneously. About 20% of myosins were replaced within 10 min, while 70% of myosins were exchanged over 10 h with symmetrical and biphasic alteration of myosin release and insertion rates. Next, a fluorescence pulse-chase assay was conducted to investigate whether myosin is incorporated into specific regions in the thick filament. Newly synthesized myosin was located at the tip of the thick filament rather than the center in the first 7 min of pulse-chase labeling and was observed in the remainder of the thick filament by 30 min. These results suggest that the myosin replacement rate differs depending on the regions of the thick filament. We concluded that myosin release and insertion occur concurrently and that myosin is more frequently exchanged at the tip of the thick filament.