The influence of semicrystalline order on the second-harmonic generation (SHG) efficiency in the anisotropic bands of Drosophila melanogaster sarcomeres from larval and adult muscle has been investigated. Differences in the semicrystalline order were obtained by using wild-type and mutant strains containing different amounts of headless myosin. The reduction in semicrystalline order without altering the chemical composition of myofibrils was achieved by observing highly stretched sarcomeres and by inducing a loss of viability in myocytes. In all cases the reduction of semicrystalline order in anisotropic bands of myocytes resulted in a substantial decrease in SHG. Second-harmonic imaging during periodic contractions of myocytes revealed higher intensities when sarcomeres were in the relaxed state compared with the contracted state. This study demonstrates that an ordered semicrystalline arrangement of anisotropic bands plays a determining role in the efficiency of SHG in myocytes.