Using a modified equilibrium dialysis cell the rate of diffusion of butyrate through pig colonic mucus has been compared with that through other gels and unstirred layers. Relative diffusion coefficients were calculated for each layer. Layers of 8% polyacrylamide, and of caecal, mid-colonic and terminal colonic mucus, had coefficients that were 50-60% of the apparent free diffusion coefficient for butyrate, determined using layers made up of Millipore filters alone. The apparent free diffusion coefficients for butyrate (layers of agarose or filters) were 70% of previously determined values in the literature. This discrepancy can be explained by elements of the experimental procedure. All mucus layers differed significantly from layers of 2% agarose and Millipore filters but were not significantly different from layers of 8% polyacrylamide or from each other. Diffusion coefficients for butyrate in the mucus samples correlated with water content and carbohydrate content but had no relationship to protein content. The rate of diffusion of butyrate in colonic mucus layers was significantly reduced when compared with unstirred layers (P less than 0.05). Whether this has an effect on the butyrate supply to colonocytes in vitro and whether mucus in colonic disease behaves differently are subjects for further investigation.