The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, carbon dioxide and hydrogen gas (H2), obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is classified as a Group 4 hydrogenase and is encoded by a 14-gene operon that contains hydrogenase and Na+/H+ antiporter modules. Herein a His-tagged 4-subunit cytoplasmic subcomplex of MBH (C-MBH) was engineered and expressed in P. furiosus by differential transcription of the MBH operon. It was purified under anaerobic conditions by affinity chromatography without detergent. Purified C-MBH had a Fe : Ni ratio of 14 : 1, similar to the predicted value of 13 : 1. The O2 sensitivities of C-MBH and the 14-subunit membrane-bound version were similar (half-lives of ∼15 h in air), but C-MBH was more thermolabile (half-lives at 90°C of 8 and 25 h, respectively). C-MBH evolved H2 with the physiological electron donor, reduced ferredoxin, optimally at 60°C. This is the first report of the engineering and characterization of a soluble catalytically active subcomplex of a membrane-bound respiratory hydrogenase.