Tarantula leg muscles in the relaxed state were rapidly frozen against a copper block cooled with liquid helium. Thin longitudinal sections of freeze-substituted specimens, both live and skinned, clearly showed the helical tracks of crossbridges on the surface of the myosin filaments, which are not preserved by conventional fixation. Fourier transforms of selected filaments showed a myosin layer line pattern, similar to that observed in X-ray diffraction patterns of intact tarantula muscle, extending to the sixth order of the 43.5 nm X-ray repeat. The phases of corresponding reflections were similar on the two sides of the meridian on the first layer line, and the crossbridge arrangement showed a line of mirror symmetry running down the center of the filament. These observations show that the number of helices (N) is even, in agreement with N = 4 determined from image analysis of negatively stained, isolated tarantula filaments (Crowther et al., J. Mol. Biol. 184, 429-439, 1985). Filtered images showed clear detail of the crossbridge helices and were similar to filtered images of negatively stained, isolated thick filaments. Thus, rapid freezing combined with freeze-substitution preserves the crossbridges in a three-dimensional arrangement approximating that occurring in vivo.