Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by alpha-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu(-1)-Ser(1) bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu(-12)-Lys(-11) bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 A degrees C and retained approximately 90 % of its original activity after incubation at 70 A degrees C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys(32)-Cys(48) and Cys(180)-Cys(183) cumulatively contributed to the thermostability of TS-GSE.