Deletion of the phosphoglucose isomerase gene, PGI1, in Saccharomyces cerevisiae leads to a phenotype for which glucose is toxic. This is related to overproduction of NADPH through the oxidative part of the pentose phosphate pathway and the incompetence of S. cerevisiae to deal with this overproduction. A similar deletion (rag2) in Kluyveromyces lactis does not lead to such a phenotype. We transformed a genomic library of K. lactis in a yeast vector to a S. cerevisiae strain with a pgi1 deletion and screened for growth on glucose. We found a gene (GDP1) which encodes a phosphorylating glyceraldehyde-3-phosphate dehydrogenase, NADP-GAPDH (EC 126.96.36.199), that accepts both NADP and NAD. This is the first report of a eukaryotic, nonplant, NADP-linked GAPDH. Presumably, operation of this enzyme in the reverse direction enabled the transformed S. cerevisiae pgi1 deletion mutant to reoxidize the excess NADPH produced when glucose catabolism was forced through the pentose pathway. On the other hand, transcription of the gene in K. lactis was upregulated during growth on D-xylose, which suggests that in K. lactis the enzyme is involved in regeneration of NADPH needed for xylose assimilation, but transcription was not detected in a rag2 mutant grown on glucose. The presence of an asparagine (Asn46 in NADP-GAPDH) instead of the conserved aspartate found in related but NAD-specific enzymes may explain the ability of NADP-GAPDH to work with NADP as well as NAD.