The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100(deg)C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protease, previously designated PfpI (P. furiosus protease I) (S. B. Halio, I. I. Blumentals, S. A. Short, B. M. Merrill, and R. M. Kelly, J. Bacteriol. 178:2605-2612, 1996). The protease contains a single subunit with a molecular mass of approximately 19 kDa and exists in at least two functional conformations, which were purified separately. The predominant form from the purification (designated PfpI-C1) is a hexamer with a molecular mass of 124 (plusmn) 6 kDa (by gel filtration) and comprises about 90% of the total activity. The minor form (designated PfpI-C2) is trimeric with a molecular mass of 59 (plusmn) 3 kDa. PfpI-C1 hydrolyzed both basic and hydrophobic residues in the P1 position, indicating trypsin- and chymotrypsin-like specificities, respectively. The temperature optimum for Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-MCA) hydrolysis was (symbl)85(deg)C both for purified PfpI-C1 and for proteolytic activity in P. furiosus cell extract. In contrast, the temperature optimum for PfpI prepared by incubating a cell extract of P. furiosus at 98(deg)C in 1% sodium dodecyl sulfate for 24 h at 95 to 100(deg)C (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1255-1262, 1990), designated PfpI-H, was (symbl)100(deg)C. Moreover, the half-life of activity of PfpI-C1 at 98(deg)C was less than 30 min, in contrast to a value of more than 33 h measured for PfpI-H. PfpI-C1 appears to be a predominant serine-type protease in cell extracts but is converted in vitro, probably in part by deamidation of Asn and Gln residues, to a more thermally stable form (PfpI-H) by prolonged heat treatment. The deamination hypothesis is supported by the differences in the measured pI values of PfpI-C1 (6.1) and PfpI-H (3.8). High levels of potassium phosphate (>0.5 mM) were found to extend the half-life of PfpI-C1 activity towards AAF-MCA by up to 2.5-fold at 90(deg)C, suggesting that compatible solutes play an important role in the in vivo function of this protease.