2,4-Dichlorophenoxyacetic acid (2,4-D) is an anionic herbicide. The purpose of the present study is to examine whether organic anion transporter 3 (Oat3; Slc22a8) is solely responsible for the uptake of 2,4-D by the isolated rat choroid plexus (CP). When expressed in LLC-PK1 cells, rOat3 was mainly localized to the basolateral membrane. Although there was no vectorial transport of 2,4-D in the control LLC-PK1 cells, expression of rOat3 increased the basal-to-apical transport of 2,4-D fourfold without affecting the transcellular transport in the opposite direction. The basal-to-apical transport of 2,4-D in rOat3-LLC was saturable with a K(m) value of 20 microM. The uptake of 2,4-D by the isolated rat CP was determined using the centrifugal filtration method. Saturable uptake of 2,4-D was observed in the isolated rat CP with a K(m) value of 22 microM. Probenecid and substrates of rOat3, such as p-aminohippurate, benzylpenicillin, and cimetidine, inhibited the uptake of 2,4-D by the isolated rat CP. Their K(i) values were comparable with those for the uptake of benzylpenicillin by the isolated rat CP, which is mainly mediated by rOat3. Furthermore, benzylpenicillin was a competitive inhibitor for the uptake of 2,4-D by the isolated rat CP. These results suggest that 2,4-D and benzylpenicillin share the same transporter for their uptake by the isolated rat CP, and rOat3 is the most likely candidate transporter.