We examined the structural changes in relaxed glycerinated rabbit psoas muscle fibers induced by modification of the myosin heads with p-phenylenedimaleimide (p-PDM), which reacts with sulfhydryls on the myosin head to cause its loss of ability to combine with actin and to hydrolyse ATP. In the longitudinal sections of both chemically fixed and quickly frozen muscle fibers, ladder-like structures, interpreted as the myosin heads extending nearly at right angles with the thick filaments, were more prominent in the p-PDM-modified fibers than in the control fibers. Fourier transform diffractograms of the longitudinal sections exhibited a distinct 14.3-nm meridional reflection, which arises from axial spacing of the myosin heads on the thick filament, in the p-PDM-modified fibers, but not in the control fibers. These results indicate that the p-PDM-modification of the myosin heads causes an increase in the regularity of myosin head arrangement on the thick filament.