Intrafiber diffusion of 3HOH, dimethyl-3H-sulfoxide (DMSO), D-14C-sorbitol, and 36Cl was measured along the longitudinal axis of the single muscle fiber (Balanus nubilus) that had been placed inside the lumen of a glass capillary at least 24 h beforehand. When the fiber contained 75% water, the mean self-diffusion coefficients (× 10−5 cm2/s) at 10 °C and at pH 7.5 were 0.908 ± 0.008 for water, 0.418 ± 0.008 for DMSO, 0.216 ± 0.005 for sorbitol, and 0.683 ± 0.006 for chloride. These diffusion coefficients in myoplasm were 0.53–0.58 times the values in pure water. Diffusions of the above were also measured in fibers with reduced water content, as low as 45% by weight. In all cases, the diffusion coefficient decreased in proportion to the reduction in the fiber water. With the aid of Wang's theory for diffusion in polyelectrolyte solutions, we have attempted to separate the "obstruction effect" from the "binding effect", both of which operate to reduce the diffusion coefficient of substances in the myoplasm. Our analysis indicates that the diffusible volume in myoplasm (75% water) for all substances is about 80% of the water volume or 65% of the fiber volume. So-called "bound" water in myoplasm is estimated to be 0.57 g water per gram dry weight.