Abstract: Most yeast hexose transporters studied so far at the molecular level mediate facilitated diffusion of glucose and fructose. Here, we report that a novel Kluyveromyces lactis gene, FRT1, encodes a proton-coupled fructose-uptake transporter. Frt1, when expressed in a Saccharomyces cerevisiae hxt null mutant strain that is unable to take up monosaccharides, restored growth on fructose. Determination of substrate specificities and kinetic parameters revealed Frt1 as a fructose transporter with a K(m) of 0.16+/-0.02 mM. Uptake of fructose was accompanied by an initial alkalization of the medium, indicating a proton-coupled uptake mechanism. Deletion of the FRT1 gene in a K. lactis strain already deleted for its RAG1 and HGT1 hexose transporter genes completely prevented uptake of and growth with fructose but not with glucose. Kinetic parameters of Frt1 in K. lactis, as assessed in a rag1 hgt1 mutant strain, were comparable with those obtained after heterologous expression in S. cerevisiae. Transcription of the FRT1 gene, which was undetectable when cells were grown in ethanol, was induced by various sugars. Our results indicate that, unlike S. cerevisiae, K. lactis exhibits proton symport systems for the uptake of hexoses, in addition to their facilitated diffusion.