We have isolated the gene encoding transaldolase from Kluyveromyces lactis (KITAL1) by screening a genomic library of this yeast using the TAL1 gene of Saccharomyces cerevisiae as a radioactive probe. The clone isolated contained an open reading frame of 1002 bp, encoding a protein with 76% identical residues in the deduced amino acid sequences as compared to Tal from S. cerevisiae. KITAL1 can complement a tal1 deletion of S. cerevisiae for enzymatic activity. The transcription start of KITAL1 was located at -69 bp relative to the ATG translation start codon. Deleting a large part of the open reading frame from the genome did not lead to any obvious phenotype. Transaldolase was not produced in such mutants as shown by immunological detection. In combination with a double null-mutant in the genes encoding the phosphofructokinase subunits in K. lactis (Klpfk1 Klpfk2 Kltal1), the cells lost their ability to grow on glucose. We take this as strong evidence that glucose is metabolized via the pentose phosphate pathway in this yeast when glycolysis is blocked. In addition, by tetrad analysis we detected a close linkage to KIPFK1 and inferred that KITAL1 is localized on chromosome I.