The thermostable esterase from the thermophilic bacterium Clostridium thermocellum DSM 1313 was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. Its molecular weight was approximately 35 kDa according to 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The enzyme exhibited the highest specific activity with p-nitrophenyl butyrate (285 s(-1) mM(-1)). The activity of the esterase was greatest at 65 A degrees C, and the esterase maintained residual activity levels of 70 and 50 % after 3 h incubation at 65 and 70 A degrees C, respectively. Its activity was optimal at pH 7.0, was enhanced in the presence of Ca2+ and Mg2+, and was inhibited by Ni2+ and Cu2+. The addition of surfactants, such as Tween-20, Tween-80, Triton X-100, and SDS, at concentrations of 5 % (v/v) significantly inhibited the lipolytic action of the esterase. Enzyme activity was relatively stable in 10 % methanol, and 50 % residual activity was seen in 10 % DMSO, demonstrating its potential in biodiesel production and industrial applications.