Abstract It has been reported that the hyperthermophilic archaeon, Methanococcus jannaschii, possesses two FKBP (FK506 binding protein) genes in the genome, one being 26 kDa FKBP (long-type FKBP) and the other, 18 kDa FKBP (short-type FKBP). FKBP is a family of peptidyl–prolyl cis-trans isomerases (PPIases). In order to clarify the difference between their roles in archaeal cells, they were expressed in Escherichia coli, and their PPIase and chaperone-like protein-folding activities were investigated. The catalytic efficiency of the PPIase activity of the long-type FKBP was significantly lower than that of short-type FKBP (less than 1/1000) which is comparable to that of human FKBP12. Both FKBPs showed chaperone-like protein-folding activity to enhance the refolding yield of an unfolded protein ( Thermoplasma citrate synthase) in vitro. The chaperone-like protein-folding activity of the short type was higher than that of the long type. While the intracellular content of long-type FKBP in M. jannaschii tended to increase, that of short-type FKBP obviously decreased at growth temperatures higher than the optimum of 85 °C. In Pyrococcus horikoshii, another hyperthermophilic archaeon, the intracellular content of long-type FKBP did not change with temperature (80–102 °C). These results suggest that long-type FKBP functions at any temperature in the cells as a chaperone to maintain the folding states of intracellular proteins. On the other hand, short-type FKBP may be required at lower temperatures. Peptidyl–prolyl cis-trans isomerization is known to be a rate-limiting step in protein-folding and is slower at low temperature. Since the PPIase activity of short-type FKBP was much stronger than that of the long type, it may be required to accelerate the folding of intracellular proteins and for the hyperthermophilic cell to live at low growth temperatures.