Ornithine carbamoyltransferase (OTCase) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is a 400 +/- 20-kDa polymer of a 35-kDa subunit, in keeping with the corresponding gene sequence [Roovers, M., Hethke, C., Legrain, C., Thomm, M. & Glansdorff, N. (1997) Isolation of the gene encoding Pyrococcus furiosus ornithine cabamoyltransferase and study of its expression profile in vivo and in vitro, Eur. J. Biochem. 247, 1038-1045]. In contrast with the dodecameric catabolic OTCase of Pseudomonas aeruginosa, P. furiosus OTCase exhibits no substrate cooperativity. In keeping with other data discussed in the text, this suggests that the enzyme serves an anabolic function. Half-life estimates for the purified enzyme ranged over 21-65 min at 100 degrees C according to the experimental conditions and reached several hours in the presence of ornithine and phosphate. The stability was not markedly influenced by the protein concentration. Whereas comparative examination of OTCase sequences did not point to any outstanding feature possibly related to thermophily, modelling the enzyme on the X-ray structure of P. aeruginosa OTCase (constituted by four trimers assembled in a tetrahedral manner) suggests that the molecule is stabilized, at least in part, by a set of hydrophobic interactions at the interfaces between the trimers. The comparison between P. aeruginosa and P. furiosus OTCases suggests that two different properties, allostery and thermostability, have been engineered starting from a similar quaternary structure of high internal symmetry. Recombinant P. furiosus OTCase synthesised by Escherichia coli proved less stable than the native enzyme. In Saccharomyces cerevisiae, however, an enzyme apparently identical to the native one could be obtained.