A genomic DNA fragment, encoding a thermotolerant beta-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5 kDa protein (by SDS-PAGE) encoded by 1.341 kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-beta-D-glucopyranoside were 2.8 mM and 42.7 mmol min(-1) mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.