The branched chain amino acid biosynthesis pathway is responsible for the production of L-valine, L-leucine and L-isoleucine in plants, fungi and bacteria, while animals do not have this set of enzymes. Experiments including gene knockout studies have confirmed that the activity of this pathway is essential to the survival of many of these organisms. The enzymes of this pathway are therefore potential targets for biocide discovery. Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), the target of over 50 commercial herbicides, is the first common enzyme of the pathway. It consists of two types of subunits, one catalytic (CSU) and the other regulatory (RSU). The RSU enhances CSU activity and allows feedback inhibition by the branched chain amino acids. While structures of the CSU and RSU were separately resolved, knowledge of the interactions that occurs between the two subunits has been impeded by the lack of a three-dimensional structure for any AHAS complex. Here, the three-dimensional structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana AHAS complex has been determined. The structures show that the RSU forms a tetrameric core to which four CSU dimers are attached, taking the shape of a ‘Maltese cross’. These structures reveal a pair of salt bridges between the CSU and RSU, which guides the movement of the CSU during catalysis resulting in increased CSU activity. The structure of A. thaliana AHAS in complex with L-valine showed that the binding of L-valine to the RSU causes a series of conformational changes, including the disruption of the salt bridge, that result in feedback inhibition of AHAS activity. In addition, a similar structure was also observed for the Mycobacterium tuberculosis AHAS complex, suggesting conservation of the AHAS architecture across kingdoms.