Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants.De novobiosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family ofArabidopsispurine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of aSaccharomyces cerevisiaemutant strain impaired in bothde novobiosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well asin planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in apup1mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining thepup1mutant with a vitamin B6de novobiosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.