SUMMARYFlightin is a multiply phosphorylated, myosin-binding protein found specifically in indirect flight muscles (IFM) of Drosophila. A null mutation in the flightin gene (fln0) compromises thick filament assembly and muscle integrity resulting in muscle degeneration and lost of flight ability. Using P-element-mediated transformation with the full-length flightin gene driven by the Actin88F promoter,we have achieved rescue of all fln0-related ultrastructural and functional defects of the IFM. Transgenic P{fln+}fln0 `rescued' flies have fewer thick filaments per myofbril than wild-type flies (782±13 vs945±9) but have otherwise normal IFM. Transgenic P{fln+}fln+ `tetraploid' flies have a normal number of thick filaments. The flightin protein levels in both transgenic strains are similar to wild type. By contrast, flightin levels are reduced in a myosin heavy chain tetraploid strain that produces excess myosin and excess thick filaments. These results suggest that regulation of flightin protein level is independent of gene copy number and that the number of thick filaments assembled per myofibril is influenced independently by myosin and flightin expression. We measured mechanical properties of IFM skinned fibers by sinusoidal analysis and found no significant differences in active viscoelastic properties of flightin-rescued and tetraploid transgenic flies vs wild type. The ability of the fln+transgene to overcome deficits in dynamic stiffness and power output in fln0 suggest that the flightin protein contributes directly to fiber stiffness and stretch activation. However, flight parameters at maximum locomotor capacity, measured in a virtual reality flight simulator,are slightly compromised for both transgenic strains. P{fln+}fln0 and P{fln+}fln+ flies generated enough flight force to sustain hovering flight but showed reduced capability to produce forces in excess of hovering flight force. Both strains showed reductions in stroke frequency but only P{fln+}fln+ showed reductions in stroke amplitude. Muscle and aerodynamic efficiency are similar among the two transgenic strains and wild type. These results illustrate the importance of flightin in flight muscle development and function.