The assembly of striated muscle myosin into thick filaments of precise and regular length requires the assistance of accessory proteins. Drosophila indirect flight muscle (IFM) contain flightin, a 20-kDa protein that has been shown to be essential for flight, for maintenance of sarcomeric integrity in active muscle, and informative in length determination of thick filaments during IFM development. Additionally, a point mutation in the myosin rod (Mhc(13)) negates flightin accumulation in the IFM in vivo. The manner in which flightin interacts with thick filaments is not known. Here, two different solid-state binding assays demonstrate that flightin binds to myosin and to a recombinant fragment of the myosin rod that include the COOH-terminal 600 amino acids (zone 19 to tail piece). The interaction of flightin and myosin is abolished by the single amino acid substitution in Mhc(13) at position 1e of zone 27 of the rod (residue 1554). The molar ratio of flightin to myosin is approx 1 : 1 to 1 : 2. Thus, the instability of thick filaments seen in vivo in the absence of flightin suggests that the flightin myosin interaction is critical for maintaining sarcomere integrity in active muscle.