The transfer of the cationic drugs, pralidoxime (PAM) and tetraethylammonium, and anionic ampicillin from the mucosal-to-serosal sides of everted rat jejunal sacs is enhanced by mucosal hypertonicity. PAM uptake, which is proportional to initial mucosal concentrations up to 2.3 mM, is enhanced by mucosal hypertonicity due to addition of sodium, potassium, lithium and choline chloride, sodium sulfate, and the nonionic solutes, urea, sucrose, and mannitol. Bicarbonate, Tris, or phosphate buffer and the presence of magnesium and calcium do not affect this hypertonicity-induced acceleration of PAM passage. Serosal osmolality has no effect on transfer and mucosal hypertonicity is equally effective in the presence and absence of a transmural osmotic gradient. This observation and minimal changes in the concentration of inulin placed in the sacs suggest that fluid shifts and solvent drag are not responsible for the enhanced mucosal-to-serosal transfer of PAM from hypertonic buffer. Mucosal hypertonicity at 450 mosmol/kg causes reversible enhancement of PAM transfer, whereas the effect of 600 mosmol/kg cannot be reversed by replacing the tissue in isotonic buffer. The effect of osmotic manipulation on PAM transfer across the intestine thus differs from its effect on the passage of other ionized species and drugs across other epithelia.