Objective: The proton-coupled peptide transporter, hPEPT1, is situated in the apical membrane of small intestinal enterocytes. hPEPT1 transports di- and tripeptides, drug substances such as b-lactam antibiotics and d-aminolevulinic acid from the intestinal lumen into the enterocytes. The aim of the present study was to investigate if ibuprofen binds to hPEPT1 and to evaluate if drug-drug interactions could be a consequence. Methods: Inhibition by ibuprofen of hPEPT1- mediated [¹⁴C]Gly-Sar uptake was determined in MDCK/hPEPT1 cells. The cellular toxicity of ibuprofen was investigated using a 3- (4, 5-dimethylthiazol-2-y1)2, 5-diphenyl tetrazolium bromide (MTT) assay. [³H] ibuprofen uptake into MDCK/hPEPT1 and MDCK/Mock cells was studied in the absence or presence of known hPEPT1 substrates. Results: The uptake of [¹⁴C]Gly-Sar in MDCK/hPEPT1 cells was reduced to 7±1% and 4±6% of the control uptake in the presence of 20 mM Gly-Pro or 10 mM ibuprofen, respectively. Ibuprofen elicited a concentration dependent inhibition of [¹⁴C]Gly-Sar uptake with a K_i of 0.4 mM (0.2-0.8 mM, 95 % CI). The MTT test suggested that ibuprofen was not toxic < 10 mM. The uptake of [³H]ibuprofen into MDCK/hPEPT1 and MDCK/Mock cells was however not inhibited by hPEPT1 substrates such as Gly-Pro, d- aminolevulinic acid or ampicillin. In addition, the [³H]ibuprofen uptake rates into MDCK/hPEPT1 cells and MDCK/Mock cells were similar. Thus, ibuprofen inhibits the uptake of hPEPT1 substrates without itself being transported by hPEPT1. A normal dosage of ibuprofen is 400 mg resulting in intestinal luminal concentrations of up to 8 mM, or 20 times the K_i of ibuprofen. Considerable inhibition of hPEPT1-mediated transport is thus expected. Conclusion: We have identified ibuprofen as an inhibitor of hPEPT1. Drug-drug interactions at the level of intestinal drug absorption via hPEPT1 are anticipated in dose regimens consisting of ibuprofen and hPEPT1 substrates.