The mucosa-to-serosa permeability of non-electrolytes through the stripped colonic rat mucosa was examined in a Ussing-type chamber. The permeation clearances for inulin (12-15 A radius) to erythritol (3.2 A radius) increased linearly with the increase in their free diffusion coefficients. On the other hand, the clearances of glycerol, thiourea and urea of less than 3 A radius increased in excess of what would be expected considering the above linearity. This suggests that there are large pores that do not restrict diffusive flow and small pores of 3 A radius or greater that restricted diffusion in the paracellular channel. The transcellular permeation also occurred in a tracer efflux experiment with urea preloaded in colonic stripped mucosa. It was not ruled out that the higher permeability of the nonelectrolytes less than 3 A in radius was due to a transcellular route in parallel with the paracellular one.