Sugar transport is very critical in developing an efficient and rapid conversion process of a mixture of sugars by engineered microorganisms. By using expressed sequence tag data generated for the fructophilic yeast Candida magnoliae JH110, we identified two fructose-specific transporters, CmFSY1 and CmFFZ1, which show high homology with known fructose transporters of other yeasts. The CmFSY1 and CmFFZ1 genes harbor no introns and encode proteins of 574 and 582 amino acids, respectively. Heterologous expression of the two fructose-specific transporter genes in a Saccharomyces cerevisiae, which is unable to utilize hexoses, revealed that both transporters are functionally expressed and specifically transport fructose. These results were further corroborated by kinetic analysis of the fructose transport that showed that CmFsy1p is a high-affinity fructose-proton symporter with low capacity (K (M) = 0.13 +/- 0.01 mM, V (max) = 2.1 +/- 0.3 mmol h(-1) [gdw](-1)) and that CmFfz1p is a low-affinity fructose-specific facilitator with high capacity (K (M) = 105 +/- 12 mM, V (max) = 8.6 +/- 0.7 mmol h(-1) [gdw](-1)). These fructose-specific transporters can be used for improving fructose transport in engineered microorganisms for the production of biofuels and chemicals from fructose-containing feedstock.