Tk-subtilisin, a hyperthermostable subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca(2+) ions. Four of them (Ca2-Ca5) are responsible for folding of Tk-subtilisin. In this study, to clarify the role of the other three Ca(2+) ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/ΔCa1), Ca6 ion (Pro-TKS/ΔCa6), and Ca7 ion (Pro-TKS/ΔCa7), and their active site mutants (Pro-S324A/ΔCa1, Pro-S324A/ΔCa6, and Pro-S324A/ΔCa7, respectively). Pro-TKS/ΔCa6 and Pro-TKS/ΔCa7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/ΔCa1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-subtilisin/ΔCa1 was determined to be 50% of that of Tk-subtilisin using the refolded protein. This result suggests that the Ca1 ion is required for the maximal activity of Tk-subtilisin. The refolding rates of all Pro-S324A derivatives were comparable to that of Pro-S324A (active site mutant of Pro-TKS), indicating that these Ca(2+) ions are not needed for folding of Tk-subtilisin. The stabilities of Pro-S324A/ΔCa1 and Pro-S324A/ΔCa6 were decreased by 26.6 and 11.7 °C, respectively, in T(m) compared to that of Pro-S324A. The half-lives of Tk-subtilisin/ΔCa6 and Tk-subtilisin/ΔCa7 at 95 °C were 8- and 4-fold lower than that of Tk-subtilisin, respectively. These results suggest that the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin.