AbstractWe have developed recombinant Saccharomyces yeasts that can effectively co‐ferment glucose and xylose to ethanol. However, these yeasts still ferment glucose more efficiently than xylose. The transport of xylose could be one of the steps limiting the fermentation of xylose. In this study, we characterized the changes in the expression pattern of the hexose transporter and related genes during co‐fermentation of glucose and xylose using one of our recombinant yeasts, Saccharomyces cerevisiae 424A(LNH‐ST). The transcription of the hexose transporter and related genes was strongly influenced by the presence of glucose; HXT1, HXT2 and HXT3 were greatly activated by glucose and HXT5, HXT7 and AGT1 were significantly repressed by glucose. We also examined the effectiveness of individual transporters encoded by HXT1, HXT2, HXT4, HXT5, HXT7 and GAL2 genes for transporting xylose during co‐fermentation of glucose and xylose in a Saccharomyces hxt° mutant (RE700A). We compared these hxt° derivatives to RE700A wild‐type strain (S. cerevisiae MC996A) where all of them contained the same xylose metabolizing genes present in our xylose‐fermenting yeasts such as 424A(LNH‐ST). Our results showed that recombinant RE700A containing the cloned HXT7 or HXT5 were substantially more effective for fermenting xylose to ethanol. In addition, we found that the efficiency of transporters for intracellular accumulation of xylose was as follows: HXT7 > HXT5 > GAL2 > WT > HXT1 > HXT4 > > > RE700A. Furthermore, we provided evidence that the Saccharomyces galactose transporter system could be a highly effective xylose transporter. The information reported here should be of great importance for improving the Saccharomyces yeast transport of xylose. Copyright © 2004 John Wiley & Sons, Ltd.