A detailed investigation of the spin-diffusion coefficient Ds of water protons in skeletal muscle has been studied by pulsed nuclear magnetic resonance (NMR) methods. Skeletal muscles of mature male rats were placed in a sample holder in which the diffusion coefficient (Ds) of water could be determined as a function of fiber axis theta. The value of Ds(theta) was determined for theta = 0 degrees, 45 degrees, and 90 degrees. The measured anisotropy Ds(O)/Ds(90) was 1.39, and the value of Ds(O) was 1.39 X 10(-5) cm2/sec. 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 three major conclusions: (1) interpretations in which the reduction in Ds is ascribed to the effect of geometrical obstructions on the diffusion of "free" water are ruled out; (2) there is a large fraction of the cellular water bound or otherwise associated with the proteins in such a way that its diffusion coefficient is substantially reduced; and (3) cellular water cannot be considered to be equivalent to a dilute solution.