In the present study, we have investigated ibuprofen transport in renal cell cultures with the aim of characterizing a recently proposed ibuprofen transporter. We here show that hyperosmolarity upregulates an ibuprofen transporter in dog renal MDCK I cells via increased mRNA and protein synthesis, while the transporter was not upregulated by hyperosmolarity in porcine and rat renal cell lines. The transport of ibuprofen in MDCK I cells was inhibited by ibuprofen metabolites such as 1- and 3-hydroxyibuprofen, and to a lesser extent by 2-hydroxyibuprofen. Other non-steroidal anti-inflammatory drugs such as naproxen, ketoprofen, flurbiprofen, diclofenac, and piroxicam also inhibited ibuprofen transport while no effect was observed from acetylsalicylic acid. The transporter was functionally detected in both the apical and basolateral membranes of MDCK 1 cells, and may control intracellular concentrations of ibuprofen.