Acetate kinase (EC 2.7.2.1) is involved in the wasteful production of acetate during conversion of cellulose to ethanol by Clostridium thermocellum. The properties of this enzyme activity in C. thermocellum cell extracts were determined. Optimum enzyme activity was at 60 degrees C and between pH 7.5 and 9.0. In the presence of air, acetate kinase was stable to temperatures up to 60 degrees C, retaining 90% activity after 2 h, and was inactivated rapidly at higher temperatures. The enzyme exhibited a wide range of stability to pH (5.0-9.0) when incubated at 50 degrees C for 2 h. As with other acetate kinases, a divalent cation, such as Mg2+, was required for enzyme activity. Optimum activity was observed at 20 mM MgCl2 when ATP was held constant at 10 mM. Acetate kinase activity was adversely affected by KCl, a salt commonly used in ion-exchange or affinity chromatography, with 0.3 M KCl inhibiting by 50%. These results will be important in optimizing the direct microbial conversion process of cellulose to ethanol using C. thermocellum in coculture with Clostridium thermosaccharolyticum.