We have studied the structure and function of muscle fibers in which tropomyosin stoichiometry has been reduced by genetic mutation. We used a Drosophila melanogaster flightless mutant Ifm(3)3 and a genetic cross of this mutant with wild type flies to achieve a gradation of tropomyosin gene dosage. We measured the flight ability and wingbeat frequency of the live insects and the ultrastructure and mechanochemistry of isolated single flight muscle fibers. Flight ability is impaired when tropomyosin gene dosage is reduced. Wingbeat frequency also depends upon gene dosage as well as the severity of myofilament lattice disruption and the number of myofilaments in the organized core of the myofibrils. A reduction in number of myofilaments appears to result in a reduction in active muscle stiffness without resulting in an appreciable change in kinetics of force production. Ifm(3)3 is trapped in a relaxed state and cannot generate active force. However, tight-binding rigor cross-bridges are able to form; in the absence of ATP, Ifm(3)3 muscle fibers have high stiffness and force.