Myosin regulatory light chain is phosphorylated by myosin light chain kinase at conserved serine and threonine residues in a number of species. Phosphorylation of myosin regulatory light chain regulates smooth muscle contraction, but appears to have a modulatory role in striated muscle contraction. We assessed the in vivo role of myosin regulatory light chain phosphorylation in the striated muscles of Drosophila melanogaster by substituting alanine at each or both conserved myosin light chain kinase-dependent phosphorylation sites, serine 66 and serine 67. We report here that myosin light chain kinase-dependent phosphorylation is not required for myofibrillogenesis or for the development of maximal isometric force in indirect flight muscles. However, mutants with substitutions at the major phosphorylation site (serine 66) or with the double substitutions had reduced power output in isolated flight muscle fibres and reduced flight ability, showing that myosin regulatory light chain phosphorylation is a key determinant of the stretch activation response in Drosophila.