Chemically demembranated bundles of fibers from tarantula leg muscle were rapidly frozen in the relaxed state and freeze-substituted in the presence of tannic acid. Electron micrographs of thin transverse sections of freeze-substituted specimens frequently showed four clear, regularly organized projections (crossbridges) protruding from the backbones of the myosin filaments and partially wrapping around the filament surface. The rotational power spectra of individual filaments showed a peak at N = 4. Alignment and averaging of the images using correlation methods confirmed the fourfold symmetry and the slewed configuration of the crossbridges on the filament surface. These observations directly reveal essential features of the low-resolution three-dimensional helical reconstruction of negatively stained tarantula filaments calculated previously (R. A. Crowther, R. Padrón, and R. Craig, 1985, J. Mol. Biol. 184, 429-439).