We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipitis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the nonoxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-l,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae X Y L 1 / X Y L 2 transformants.