Complex II from the thermoacidophilic archaeon Acidianus ambivalens, an archetype of an emerging class of succinate dehydrogenases (SDH), was extracted from intact membranes and purified to homogeneity. The complex contains one molecule of covalently bound FAD and 10 Fe atoms. EPR studies showed that the complex contains the canonical centres S1 ([2Fe-2S]2+/1+) and S2 ([4Fe-4S]+2/+1) but lacks centre S3 ([3Fe-4S]+1/0); these observations agree with the fact that the iron-sulfur subunit contains an extra cysteine that may allow the binding of a new centre, most probably a tetranuclear one. Succinate-driven oxygen consumption is observed in intact membranes indicating that in vivo, complex II operates as a succinate:quinone oxidoreductase, despite missing the typical anchor domain subunits. The pure complex was found to contain bound caldariella quinone, the enzyme physiological partner. An alternative membrane anchoring for this new type of SDHs, based on the amphipathic nature of the putative helices found in SdhC, is suggested.