Lignocellulosic biomass, which contains large amounts of glucose and xylose, is the new ideal feedstock for ethanol production used as renewable liquid fuel for transportation. The naturally occurring Saccharomyces yeasts traditionally used for industrial ethanol production are unable to ferment xylose. We have successfully developed genetically engineered Saccharomyces yeasts that can effectively co-ferment both glucose and xylose simultaneously to ethanol. Our engineered yeast contains three xylose metabolizing genes, the xylose reductase (XR), xylitol dehydrogenase (XD) and xylulokinase (XK) genes, fused to glycolytic promoters, on high copy plasmids or integrated into the yeast chromosome in multiple copies. Although our glucose/xylose co-fermenting yeasts are currently the most effective yeast for producing ethanol from cellulosic biomass, they still utilize glucose more efficiently than xylose. We believe that carefully analyzing gene expression during co-fermentation of glucose and xylose to ethanol, using our genetically modified strains, will reveal ways to optimize xylose fermentation. In this paper, we report our results on analyzing the expression of genes in the glycolitic and alcoholic fermentation pathways using microarray technology. We also report the results on the analysis of the activities of the selected enzymes for ethanol production during co-fermentation of glucose and xylose to ethanol by one of our effective glucose/xylose co-fermenting yeasts 424A(LNH-ST). (C) 2003 Elsevier Science Inc. All rights reserved.