The anisotropy of the spin-diffusion coefficient Ds of water protons in skeletal muscle has been studied by pulsed NMR methods. The mid-portion of the tibialis anterior muscle of mature male rats was placed in a special sample holder by means of which the muscle fiber orientation theta relative to the diffusion direction could be varied over the range 0 degrees less than or equal to theta less than or equal to 90 degrees. The value of Ds(theta) was determined for theta = 0 degrees, 45 degrees, and 90 degrees. The measured anisotropy Ds(0)/Ds(90) was 1.39, and the value of Ds(0) was 1.39 X 10(-5) cm2/s. These results are interpreted within the framework of a model calculation in which the diffusion equation is solved for a regular hexagonal network similar to the actin-myosin filament network. The large anisotropy, and the large reduction in the value of Ds measured parallel to the filament axes lead to two major conclusions: (a) interpretations in which the reduction in Ds is ascribed to the effect of geometrical obstructions on the diffusion of "free" water are ruled out; and, (b) there is a large fraction of the cellular water associated with the proteins in such a way that its diffusion coefficient is substantially reduced.