Aims: Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high-yield production of enzymatic hydrogen. Methods and Results: The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli. The purified enzyme appeared to be a monomer with an estimated molecular weight of 64 center dot 9 kDa. This enzyme was found to be a dual-specificity enzyme - PGM/phosphomannomutase (PMM). Mg2+ and Mn2+ were activators. Ser144 was identified as an essential catalytic residue through site-directed mutagenesis. The k(cat) and K-m of PGM were 190 s-1 and 0 center dot 41 mmol l-1 on glucose-1-phosphate and 59 s-1 and 0 center dot 44 mmol l-1 on mannose-1-phosphate, respectively, at 60 degrees C. Thermostability of PGM at a low concentration (2 nmol l-1, 100 U l-1) was enhanced by 12-fold (i.e. t(1/2) = 72 h) at 60 degrees C with addition of bovine serum albumin, Triton X-100, Mg2+and Mn2+. Conclusions: The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported. Significance and Impact of the Study: This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).