Contraction in striated muscles is regulated by Ca2+-dependent movement of tropomyosin-troponin on thin filaments. Interactions of charged amino acid residues between the surfaces of tropomyosin and actin are believed to play an integral role in this steric mechanism by influencing the position of tropomyosin on the filaments. To investigate this possibility further, thin filaments were isolated from troponin-regulated, indirect flight muscles of Drosophila mutants that express actin with an amino acid charge reversal at residue 93 located at the interface between actin subdomains 1 and 2, in which a lysine residue is substituted for a glutamic acid. Electron microscopy and 3D helical reconstruction were employed to evaluate the structural effects of the mutation. In the absence of Ca2+, tropomyosin was in a position that blocked the myosin-binding sites on actin, as previously found with wild-type filaments. However, in the presence of Ca2+, tropomyosin position in the mutant filaments was much more variable than in the wild-type ones. In most cases (approximately 60%), tropomyosin remained in the blocking position despite the presence of Ca2+, failing to undergo a normal Ca2+-induced change in position. Thus, switching of a negative to a positive charge at position 93 on actin may stabilize negatively charged tropomyosin in the Ca2+-free state regardless of Ca2+ levels, an alteration that, in turn, is likely to interfere with steric regulation and consequently muscle activation. These results highlight the importance of actin's surface charges in determining the distribution of tropomyosin positions on thin filaments derived from troponin-regulated striated muscles.