X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2(Delta2-46)) or disruption of the phosphorylation sites by substituting alanines (Dmlc2(S66A, S67A)) decreased the equatorial intensity ratio (I(20)/I(10)), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2(S66A, S67A)), but not N-terminal extension truncation (Dmlc2(Delta2-46)), decreased the 14.5nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2(S66A, S67A)) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2(Delta2-46; S66A, S67A)), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a "tether" to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.