Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 A and 2.5 A, respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H2O)(6)2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.