Cyclodextrin glycosyltransferase (CGTase) from the thermophilic anaerobe Thermoanaerobacter sp. 5K was purified and characterized. The enzyme was purified with ammonium sulfate precipitation followed by α-CD-bound, epoxy-activated Sepharose 6B affinity chromatography. Molecular weight of the purified enzyme was 70.6 kDa. The enzyme had optimal activity at 80-90°C and retained greater than 90% activity between 75°C and 95°C. Optimal pH activity was observed at 7.0, with at least 50% activity between pH 4.0 and 9.0. It was highly stable at elevated temperature, with no loss of activity after incubation at 80°C for 4 hours or at 90°C for 30 min. Km and Vmax values were 0.222 mg/mL and 0.206 mg β-CD/mL/min, respectively, with soluble starch. Amino acid composition of the enzyme was deduced from the sequence of the cloned CGTase gene. The mature enzyme has a deduced molecular weight of 75.63 kDa and contains residues conserved in the CGTase class of amylase enzymes.