Intrafiber diffusion of 22Na and 14C-sorbitol was measured along the longitudinal axis of a single muscle fiber which had been placed inside the lumen of a glass capillary at least 24 h beforehand. Compared to their diffusion in 1% agar, the self-diffusion coefficients of 22Na at 10 °C (0.438 × 10−5 cm2/s) and 14C-sorbitol (0.216 × 10−5 cm2/s) were reduced by a factor of 2 when intrafiber pH was 7.5. However, the theoretical and experimental concentration–distance profile curves only matched for 14C-sorbitol but not for 22Na. In the latter case, the experimental curves were consistently higher than the theoretical curve indicating an excess accumulation of 22Na at all x values (axis distance). This Na accumulation was found to be highly dependent on the pH inside the fiber. From pH 10.1 to 5.2, the fraction of excess Na (excess 22Na per total 22Na at a given x) decreased from 0.34 to zero. We interpret this excess Na to be a measure of the amount of myoplasmic Na which is acting as counterion for the fixed negative charge on the contractile filaments.