Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Ralstonia eutropha JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 μM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated tfdK, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of the tfdK gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A tfdA mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.