In skeletal muscle, the myosin molecule contains two sets of noncovalently attached low molecular weight proteins, the regulatory (RLC) and essential (ELC) light chains. To assess the functional and developmental significance of the fast skeletal isoform of the RLC (RLC-f), the murine fast skeletal RLC gene (Mylpf) was disrupted by homologous recombination. Heterozygotes containing an intronic neo cassette (RLC-/+) had approximately one-half of the amount of the RLC-f mRNA compared to wild-type (WT) mice but their muscles were histologically normal in both adults and neonates. In contrast, homozygous mice (RLC-/-) had no RLC-f mRNA or protein and completely lacked both fast and slow skeletal muscle. This was likely due to interference with mRNA processing in the presence of the neo cassette. These RLC-f null mice died immediately after birth, presumably due to respiratory failure since their diaphragms lacked skeletal muscle. The body weight of newborn RLC-f null mice was decreased 30% compared to heterozygous or WT newborn mice. The lack of skeletal muscle formation in the null mice did not affect the development of other organs including the heart. In addition, we found that WT mice did not express the ventricular/slow skeletal RLC isoform (RLC-v/s) until after birth, while it was expressed normally in the embryonic heart. The lack of skeletal muscle formation observed in RLC-f null mice indicates the total dependence of skeletal muscle development on the presence of RLC-f during embryogenesis. This observation, along with the normal function of the RLC-v/s in the heart, implicates a coupled, diverse pathway for RLC-v/s and RLC-f during embryogenesis, where RLC-v/s is responsible for heart development and RLC-f is necessary for skeletal muscle formation. In conclusion, in this study we demonstrate that the Mylpf gene is critically important for fast and slow skeletal muscle development.