AbstractSaccharomyces cerevisiae mutants, in which open reading frames (ORFs) displaying similarity to the aldo‐keto reductase GRE3 gene have been deleted, were investigated regarding their ability to utilize xylose and arabinose. Reduced xylitol formation from D‐xylose in gre3 mutants of S. cerevisiae suggests that Gre3p is the major D‐xylose‐reducing enzyme in S. cerevisiae. Cell extracts from the gre3 deletion mutant showed no detectable xylose reductase activity. Decreased arabitol formation from L‐arabinose indicates that Gre3p, Ypr1p and the protein encoded by YJR096w are the major arabinose reducers in S. cerevisiae. The ypr1 deletion mutant showed the lowest specific L‐arabinose reductase activity in cell extracts, 3.5 mU/mg protein compared with 7.4 mU/mg protein for the parental strain with no deletions, and the lowest rate of arabitol formation in vivo. In another set of S. cerevisiae strains, the same ORFs were overexpressed. Increased xylose and arabinose reductase activity was observed in cell extracts for S. cerevisiae overexpressing the GRE3, YPR1 and YJR096w genes. These results, in combination with those obtained with the deletion mutants, suggest that Gre3p, Ypr1p and the protein encoded by YJR096w are capable of xylose and arabinose reduction in S. cerevisiae. Both the D‐xylose reductase and the L‐arabinose reductase activities exclusively used NADPH as co‐factor. Copyright © 2002 John Wiley & Sons, Ltd.