The hyperthermophilic archaeonPyrococcus furiosusis a strict anaerobe. It is therefore not expected to use the oxidative tricarboxylic acid (TCA) cycle for energy transduction. Nonetheless, its genome encodes more putative TCA cycle enzymes than the closely relatedPyrococcus horikoshiiandPyrococcus abyssi, including an aconitase (PF0201). Furthermore, a two-subunit fumarase (PF1755 and PF1754) is encoded on thePyr. furiosusgenome. In the present study, these three genes were heterologously overexpressed inEscherichia colito enable characterization of the enzymes. PF1755 and PF1754 were shown to form a [4Fe–4S]-cluster-containing heterodimeric enzyme, able to catalyse the reversible hydratation of fumarate. The aconitase PF0201 also contained an Fe–S cluster, and catalysed the conversion from citrate to isocitrate. The fumarase belongs to the class of two-subunit, [4Fe–4S]-cluster-containing fumarate hydratases exemplified by MmcBC fromPelotomaculum thermopropionicum; the aconitase belongs to the aconitase A family. Aconitase probably plays a role in amino acid synthesis when the organism grows on carbohydrates. However, the function of the seemingly metabolically isolated fumarase inPyr. furiosushas yet to be established.