Xylose reductase gene of Pichia stipitis (XYL1) and aldose reductase of Saccharomyces cerevisiae (GRE3) were expressed in S. cerevisiae to explore the xylitol production patterns in batch and fed-batch cultures. Although glucose utilization and ethanol formation of the two recombinant strains were not different in batch cultures, the xylitol productivity of the strain containing the S. cerevisiae GRE3 gene was 50% of that of the strain harboring the XYL1 gene of P stipitis. Such a difference in xylitol productivity was confirmed in fed-batch cultures using ethanol as a cosubstrate for regeneration of NAD(P)H. S. cerevisiae GRE3 gene product showed a strong preference to NADPH, while the degrees of cofactor specificity of R stipitis gene for both NADPH and NADH were almost identical. Similar amounts of xylose reductase were expressed in both recombinant strains, but a strict preference to NADPH in the S. cerevisiae with the GRE3 gene limited cofactor availability for xylose conversion and concomitantly resulted in lower xylitol productivity compared with the recombinant strain containing the R stipitis XYL1 gene whose product exhibited almost the same cofactor specificity to NADPH and NADH.