The zwf gene encoding glucose 6-phosphate dehydrogenase (G6PD, EC.1.1.1.49) from Enterococcus mundtii CRL35 was cloned as a 4921 bp EcoRI fragment and analyzed. The predicted zwf gene product consists of 506 residues with a molecular mass of 58.4 kDa, and is fully active in Escherichia coli as demonstrated by its heterologous expression in the zwf-negative mutant E. coli Su294. It shows a high degree of sequence identity (40-60%) to G6PDs described in other bacteria. Upstream of the zwf gene, a homolog of the DtxR family was identified (ORF D). Analysis of the 5' sequence of ORF D revealed a potential promoter sequence, which would suggest the presence of an operon-like structure between ORF D and the zwf gene. Finally, it was found that Fe2+ stop levels have an important role as a modulator of G6PD activity. This is the first report of this type of regulation of G6PD activity. A possible involvement in oxidative stress is discussed.