Methanobacterium thermoautotrophicum (strain Marburg), which grows autotrophically on H2 and CO2, was found to contain 2-oxoisovalerate oxidoreductase (Vor) and indolepyruvate oxidoreductase flor) besides pyruvate oxidoreductase (For) and 2-oxoglutarate oxidoreductase (Kor). So far, Vor and lor have only been detected in peptide-utilizing hyperthermophilic Archaea. The four 2-oxoacid oxidoreductases were purified and characterized with respect to their subunit composition, N-terminal amino acid sequences, and catalytic properties. For and Kor were composed of four different subunits, Vor was composed of three different subunits, and Ior of two different subunits. Comparisons of the N-terminal amino acid sequences revealed that the four enzymes are structurally related to each other and to the respective enzymes from Pyrococcus and Thermococcus sp. Vor from M. thermoautotrophicum differed from Vor from Pyrococcus furiosus in being composed of only three instead of four different subunits. Evidence is presented that in the autotrophic methanogen the four 2-oxoacid oxidoreductases have anabolic functions, Vor and Ior being involved in the biosynthesis of amino acids from fatty acids taken up from the growth medium, as shown by 14C-labelling studies.