The hyperthermophilic archaeon Thermococcus kodakaraensis possesses four prefoldin genes encoding two alpha subunits (pfdA and pfdC) and two beta subunits (pfdB and pfdD) of prefoldins on the genome. pfdC and pfdD are unique genes whose orthologues are not found in Pyrococcus spp., whereas pfdA and pfdB are commonly found in both Thermococcus and Pyrococcus spp. The pfdA and pfdB are located at different loci, and pfdC and pfdD were tandemly arranged on the genome. Immunoprecipitation experiments using specific antisera, anti-PfdB and anti-PfdD, revealed that PfdB and PfdD make a complex only with PfdA and PfdC, respectively. Both PfdA/PfdB and PfdC/PfdD complexes obtained as recombinant forms showed inhibitory activity against the thermal aggregation of citrate synthase. Immunoblot experiments indicated that the PfdA/PfdB complex was expressed at all examined temperatures; however, the PfdC/PfdD complex was specifically expressed under heat-stress conditions at 93 degrees C. Transcriptional analyses showed that pfdA and pfdB were transcribed at equal levels at all examined temperatures but pfdC and pfdD were transcribed at higher levels at 93 degrees C. Furthermore, pfdA and pfdB were transcribed individually, but pfdD was cotranscribed with pfdC. A typical Pyrococcus heat-shock regulator (Phr) recognition sequence was identified at the upstream region of pfdC. The transcriptional level of pfdCD was measured in a phr disruptant, showing that the pfdCD transcript in the phr disruptant was drastically increased in comparison with that of the wild type. However, the pfdCD level was also elevated at higher temperature, indicating that heat induction of PfdC/PfdD is mainly achieved by Phr derepression but that a certain degree of induction is not under Phr control. The pfdB and pfdD disruptants were then constructed, and the growth profiles were compared. At 85 degrees C cultivation, no significant difference was observed between the wild type and the pfdD disruptant; however, less growth was observed in the pfdB disruptant. At 93 degrees C, the pfdD disruptant grew less than the wild type, and the pfdB disruptant grew the least. The results suggest that the PfdA/PfdB complex plays a crucial role at all growth temperatures and the PfdC/PfdD complex contributes to survival in a high-temperature environment.