1. Mucosa-to-serosa fluxes of seven polar non-electrolytes were determined during isotonic fluid transport across the unilateral rabbit gall-bladder preparation in an attempt to estimate the contribution of the paracellular pathway to the total transepithelial water flow.2. (3)H- and (14)C-labelled non-electrolyte tracers appeared in the transported fluid at fractions (f(n)) of their mucosal concentration which were inversely related to molecular size: ethanediol, 0.80; thiourea, 0.55; glycerol, 0.16; erythritol, 0.11; mannitol, 0.05; sucrose, 0.05; inulin, 0.02. The mean volume flow rate was 78 mul. cm(-2) hr(-1).3. While the fluxes of the larger molecules were probably due to diffusion through a small but unrestricted paracellular ;shunt' permeability, the high f(n) values obtained for the smaller molecules indicate the existence of a substantial paracellular permeability restricted to molecules smaller than erythritol.4. Upper limits to the transcellular ethanediol and thiourea permeabilities, estimated from the time constants of tracer efflux from preloaded epithelial cells, were too low to account for more than a very small fraction of the transepithelial fluxes observed in the unilateral preparation.5. Comparison of the f(n) values with the predictions of a hydrodynamic model of paracellular permeation suggests that in order to account for the large fluxes of ethanediol and thiourea, considerably more than one half of the transepithelial water flow must follow the paracellular pathway.6. Following a reduction of the mucosal osmolality to 110 m-osmole kg(-1), the apparent non-electrolyte permeability of the epithelium increased steadily over a period of 4 hr. This seems to reflect an increase in the shunt permeability rather than a change in the selectivity of the restricted permeability.7. It is concluded that during isotonic fluid transport the bulk of the transepithelial water flow crossing the epithelium passes through paracellular channels of approximately 3 A radius which are probably located in the intercellular junction.