We investigated the effects of interferon-beta (IFN-beta) and IFN-gamma on the drug efflux activity of the human intestinal Caco-2 cell line, expressing the P-glycoprotein (P-gp) on the apical membrane. The cells grown on Transwell plates were pretreated with 1000U/ml IFN-beta, IFN-gamma or a combination of both for 3 days, and then the transepithelial electrical resistance (TEER) and the vectorial transport of rhodamine-123 (Rho-123) across the cell monolayers were evaluated. Exposure to IFN-gamma reduced substantially the TEER, but the effect of IFN-beta was minimal? The apparent permeability of Rho-123 in both the basolateral-to-apical and apical-to-basolateral directions was significantly increased by IFN-gamma but scarcely by IFN-beta. The combination of IFN-gamma and IFN-beta showed similar effects to IFN-gamma alone. Meanwhile, the cellular uptake of Rho-123 from the apical side was not affected by any IFN treatment. The uptake level was increased approximately three times in the presence of verapamil, a P-gp inhibitor, and the increased level was not affected by any IFN treatment, indicating that the efflux activity mediated by P-gp in the monolayers is not altered by these cytokines. Taken together, these results suggest that IFNs modulate the permeability of Caco-2 monolayer through effect on paracellular transport rather than effect on P-gp activity.