The fission yeast Schizosaccharomyces pombe is auxotrophic for biotin (vitamin H) and growth depends on biotin uptake over the plasma membrane. Here a biotin transport mutant of Saccharomyces cerevisiae is used to identify the vht1(+) gene encoding the Schizosaccharomyces pombe plasma membrane transport protein for biotin. SpVht1p belongs to the family of allantoate transporters and has only little sequence homology to the S. cerevisiae biotin transporter. Although having dissimilar primary structures, the biotin transporters in Sz. pombe and S. cerevisiae share similar biochemical properties and regulation. Like in S. cerevisiae, biotin uptake in Sz. pombe is a high-affinity process, is optimal at acidic pH values and inhibited by protonophores, indicating that SpVht1p acts as a proton-biotin symporter. Desthiobiotin, the metabolic precursor of biotin, is also imported by SpVht1p. Deletion of vht1(+) abolishes growth on low external concentrations of the vitamin, showing that vht1(+) encodes the only protein that mediates biotin uptake in Sz. pombe. Expression of vht1(+) is maximal at low external biotin concentrations, indicating that Sz. pombe can adjust the rate of biotin uptake to meet the requirement for the vitamin.