The P-glycoprotein (P-gp) efflux pump has been shown to affect drug distribution and absorption in various organs and to cause drug resistance in cancer therapy. The aim of this work was to develop a cell line to serve as a screening system for potential substrates of P-gp. This requires a cell line with high paracellular tightness, low expression of nonhuman ABC transporters, and high expression of functional human P-gp (ABCB1). The porcine intestinal epithelial cell line, IPEC-J2, was selected as a transfection host, due to its ability to form extremely high-resistance monolayers (>10,000 Ω·cm(2)) and its low endogenous expression of ABC-type efflux transporters. The IPEC-J2 cells were transfected with a plasmid that contained the sequence of the human MDR1 gene, which encodes P-gp, followed by a selection of successfully transfected cells with geneticin and puromycin. The resulting cell line, IPEC-J2 MDR1, retained its high transepithelilal resistance (>15,000 Ω·cm(2)), which translated into low permeability of the small hydrophilic tracer, mannitol (P < 10(-7) cm·s(-1)). The lipophilic compound, diazepam, displayed high permeability resulting in a dynamic range of 1500 (PDiazepam/Pmannitol) to separate high and low permeability compounds. Human P-gp was expressed predominantly in the apical membrane, as demonstrated by immunocytochemistry, Western blots, and a high efflux ratios (Pbasolateral-apical/Papical-basolateral) of known P-gp substrates. P-gp was demonstrated to be responsible for the efflux transport by substrate profiling, combined with application of P-gp and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram, and mitoxantrone were identified as P-gp substrates. Functional P-gp expression was shown to be stable through at least 10 cell passages. In conclusion, the IPEC-J2 MDR1 cell line displays high paracellular tightness combined with high expression of human P-gp and low expression of porcine ABC transporters, and it may serve as a useful tool in drug development studies.