Among various methods for structural studies of biological macromolecules, neutron scattering and diffraction have a unique feature in that the contrast between the scattering length density of the molecules and that of the solvent can be varied easily by changing the D2O content in the solvent. This "contrast variation" technique enables one to obtain information on variations of scattering length density of the molecules of interest. Here, in order to explore the possibilities of the contrast variation technique in neutron fiber diffraction, neutron diffraction measurements of skeletal muscles were performed. The neutron fiber diffraction patterns from frog sartorius muscles were measured in various D2O concentrations in the relaxed state where no tension of muscle is produced, and in the rigor state where all myosin heads of the thick filaments bind tightly to actin in the thin filaments. It was shown that in both states, there were reflections having distinct contrast matching points, indicating a variation in the scattering length density of the protein regions in the unit cell of the muscle structure. Analysis of the equatorial reflections by the two-dimensional projected scattering length density map calculations by Fourier synthesis and model calculations provided the phase information of the equatorial reflections, with which the projected scattering length density maps of the unit cell of the hexagonal filament array in both states were calculated. The analysis showed that the scattering length density of the thick filament region was higher than that of the thin filament region, and that the scattering length density of the thick filament backbone changed as muscle went from the relaxed state into the rigor state. (C) 2009 Elsevier Inc. All rights reserved.