ABSTRACT The present study investigated the simultaneous oxidation of pyruvate and amino acids during H ₂ -evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis . The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the Δ hyhBGSL strain) and the parent strain indicated that NADPH generated via H ₂ uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis . No significant change was observed by deletion of pflDA , suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of Δ porDAB and Δ vorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H ₂ yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB , respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH ₃ , suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.