Several environmental samples from Antarctica were collected and enriched to search for microorganisms with nitrilase activity. A new thermostable nitrilase from a novel hyperthermophilic archaea Pyrococcus sp. M24D13 was purified and characterized. The activity of this enzyme increased as the temperatures rise from 70 up to 85 degrees C. Its optimal activity occurred at 85 degrees C and pH 7.5. This new enzyme shows a remarkable resistance to thermal inactivation retaining more than 50% of its activity even after 8 h of incubation at 85 degrees C. In addition, this nitrilase is highly versatile demonstrating activity toward different substrates, such as benzonitrile (60 mM, aromatic nitrile) and butyronitrile (60 mM, aliphatic nitrile), with a specific activity of 3286.7 U mg(-1) of protein and 4008.2 U mg(-1) of protein, respectively. Moreover the enzyme NitM24D13 also presents cyanidase activity. The apparent Michaelis-Menten constant (K-m) and V-max of this Nitrilase for benzonitrile were 0.3 mM and 333.3 mu M min(-1), respectively, and the specificity constant (k(cat)/K-m) for benzonitrile was 2.05 x 10(5) s(-1) M-1