The rag2 mutant of Kluyveromyces lactis cannot grow on glucose when mitochondrial functions are blocked by various mitochondrial inhibitors, suggesting the presence of a defect in the fermentation pathway. The RAG2 gene has been cloned from a K. lactis genomic library by complementation of the rag2 mutation. The amino acid sequence of the RAG2 protein deduced from the nucleotide sequence of the cloned RAG2 gene shows homology to the sequences of known phosphoglucose isomerases (PGI and PHI). In vivo complementation of the pgi1 mutation in Saccharomyces cerevisiae by the cloned RAG2 gene, together with measurements of specific PGI activities and the detection of PGI proteins, confirm that the RAG2 gene of K. lactis codes for the phosphoglucose isomerase enzyme. Complete loss of PGI activity observed when the coding sequence of RAG2 was disrupted leads us to conclude that RAG2 is the only gene that codes for phosphoglucose isomerase in K. lactis. The RAG2 gene of K. lactis is expressed constitutively, independently of the growth substrates (glycolytic or gluconeogenic). Unlike the pgi1 mutants of S. cerevisiae, the K. lactis rag2 mutants can still grow on glucose, however they do not produce ethanol.