1. The transepithelial permeability in the isolated rabbit pancreas has been studied with the aid of radioactive markers added to the bathing medium. 2. After addition of these compounds in 2 mM concentration to the medium, they equilibrate within 30 min to a steady-state concentration in the secreted fluid. The latter concentrations, expressed as percent of those in the bathing medium, are: urea 100%, glycerol 90%, erythritol 95%, mannitol 60%, lactose 5%, sucrose 4% and inulin 3%. 3. Addition of 10(-5) M carbachol to the bathing medium after 60 or 90 min of incubation results in an increase of the concentrations of mannitol, lactose sucrose and inulin in the secreted fluid. Maximal concentrations, reached about 35 min after addition of the stimulant, are: mannitol 65%, lactose 31%, sucrose 23%, inulin 8%. 4. No change in the concentration of urea is observed, while the concentrations of glycerol and erythritol increase always to 100% after addition of 10(-5) M carbachol. 5. For sucrose and lactose the increase in permeability appears to be dependent on the concentration of carbachol. 6. There is no increase in the extracellular space for lactose, sucrose and inulin after incubating fragments of the rabbit pancreas with 10(-5) M carbachol. 7. Addition of atropine 5 min or more after carbachol stimulation has no effect on enzyme secretion, but markedly inhibits the increase in sucrose permeability. 8. These results indicate that: (a) the permeability of the transcellular transport route in the isolated rabbit pancreas is determined by the size of the permeating molecules, (b) this route is probably extracellular, (c) its permeability is increased by a cholinergic agent in dose-dependent fashion, (d) the increase in permeability is not caused by the enzyme secretion as such.