A method for the determination of the diffusion coefficient, D, of solutes in the separated aqueous and residual phases of 24-hour human dental plaque is described. The separated plaque components were placed in tubing (inner diameter 0.49 mm), pulsed at one end with radiotracer, and horizontal diffusion was continued at 37 or 5 °C for 5–17 h. The results indicate that the diffusion of 14C-sucrose and 14C-inulin in plaque fluid was similar to that in water, while the diffusion of 14C-starch was unexpectedly greater than in water. The D values for the three carbohydrates studied were inversely related to their molecular weights. In plaque fluid, the diffusion of ionic species, bicarbonate, acetate, lactate, butyrate as sodium salts, was less than in water. In the plaque residue, the diffusion was restricted in relation to the packing density of the residue. While the diffusion into plaque of readily fermentable carbohydrate was not restricted, that of their bacterial metabolic products was impeded, consistent with the rapid fall and slow rise in plaque pH, known to occur after exposure to fermentable carbohydrate.