The arrangement of the various components of myosin within the myofibril has been investigated by staining the thick filament with fluorescein-labeled antibodies directed against myosin and its “subunits”, heavy meromyosin S-1, rod and light chains. The specificity of the various antisera was established by immunodiffusion, quantitative precipitin analysis, and determination of ATPase activity of antibody-antigen complexes. It was found that specific antisera can promote the dissociation of the low molecular weight subunits or light chains from the rest of the myosin molecule. From the specific fluorescent staining pattern observed in the light microscope with each antiserum, it has been possible to ascertain certain features of the interactions of myosin within the myofibril. The staining pattern obtained with antibodies to light chains provides direct evidence that these subunits are a structural component of the thick filament.