We have applied three-dimensional helical reconstruction techniques to images of myosin filaments of tarantula leg muscle obtained from rapidly frozen, freeze-substituted specimens. Computed Fourier transforms of filaments selected from longitudinal sections show up to six layer lines indexing on the 43.5-nm helical repeat of myosin crossbridges. The three-dimensional reconstruction, performed after separation of overlapped Bessel functions, shows four continuous strands of density on the surface of the filament, modulated by density at 14.5-nm intervals, corresponding to the myosin heads aligned approximately along the helical strands. In transverse view, the reconstruction shows four projections and is similar in profile to myosin filaments seen in thin transverse sections of rapidly frozen muscle. The reconstruction is similar to that of negatively stained, isolated tarantula filaments except that in the latter there is an additional modulation of the helix density, which better resolves the two heads of each myosin crossbridge. Thus, the general arrangement of the myosin heads in the freeze-substituted specimens is preserved, although finer details of structure such as individual myosin heads are lost. (C) 1995 Academic Press, Inc.