ABSTRACT Methionine sulfoxide reductase (Msr) catalyzes the thioredoxin-dependent reduction and repair of methionine sulfoxide (MetO). Although Msr genes are not present in most hyperthermophile genomes, an Msr homolog encoding an MsrA-MsrB fusion protein (MsrAB _Tk ) was present on the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis . Recombinant proteins corresponding to MsrAB _Tk and the individual domains (MsrA _Tk and MsrB _Tk ) were produced, purified, and biochemically examined. MsrA _Tk and MsrB _Tk displayed strict substrate selectivity for Met- S -O and Met- R -O, respectively. MsrAB _Tk , and in particular the MsrB domain of this protein, displayed an intriguing behavior for an enzyme from a hyperthermophile. While MsrAB _Tk was relatively stable at temperatures up to 80°C (with a half-life of ∼30 min at 80°C), a 75% decrease in activity was observed after 2.5 min at 85°C, the optimal growth temperature of this archaeon. Moreover, maximal levels of MsrB activity of MsrAB _Tk were observed at the strikingly low temperature of 30°C, which also was observed for MsrB _Tk . Consistent with the low-temperature-specific biochemical properties of MsrAB _Tk , the presence of the protein was greater in T. kodakaraensis cells grown at suboptimal temperatures (60 to 70°C) and could not be detected at 80 to 90°C. We found that the amount of intracellular MsrAB _Tk protein increased with exposure to higher dissolved oxygen levels, but only at suboptimal growth temperatures. While measuring background rates of the Msr enzyme reactions, we observed significant levels of MetO reduction at high temperatures without enzyme. The occurrence of nonenzymatic MetO reduction at high temperatures may explain the specific absence of Msr homologs in most hyperthermophiles. Together with the fact that the presence of Msr in T. kodakaraensis is exceptional among the hyperthermophiles, the enzyme may represent a novel strategy for this organism to deal with low-temperature environments in which the dissolved oxygen concentrations increase.