In the previous study we cloned Pyrococcus woesei gene coding thermostable beta-galactosidase into pET30-LIC expression plasmid. The nucleotide sequence revealed that beta-galactosidase of P. woesei consists of 510 amino acids and has a molecular weight of 59,056 kDa (GenBank Accession No. AF043283). It shows 99.9% nucleotide identity to the nucleotide sequence of beta-galactosidase from Pyrococcus furiosus. We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli strain and can be easy separated by thermal precipitation of other bacterial proteins at 85 degrees C (S. Dabrowski, J, Maciunska, and J, Synowiecki, 1998, Mol. Biotechnol; 10, 217-222), In this study we presented a new expression system for producing P. woesei beta-galactosidase in Escherichia coli and one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase), The recombinant beta-galactosidase contained a polyhistidine tag at the N-terminus (20 additional amino acids) that allowed single-step isolation by Ni affinity chromatography, The enzyme was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni2+-TED-Sepharose columns. The enzyme was characterized and displayed high activity and thermostability, This bacterial expression system appears to be a good method for production of the thermostable beta-galactosidase.