Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyzes the first step in the biosynthesis of the branched-chain amino acids. In bacteria, the enzyme has a large subunit containing the catalytic machinery and a small subunit with a regulatory role. In eucaryotes, the evidence for a regulatory subunit is largely indirect and circumstantial. We investigated the possibility that the yeast open reading frame YCL009c is an AHAS small subunit. Analysis of the DNA sequence shows that it contains all the appropriate transcription, translation and regulatory signals. YCL009c was shown to be expressed in yeast and the protein localized in mitochondria where it undergoes removal of a transit peptide targeting sequence. This putative small subunit protein (ilv6) and the catalytic subunit of yeast AHAS (ilv2) were each overexpressed in Escherichia coli and purified to near homogeneity. Reconstitution studies showed that the ilv6 protein stimulates the catalytic activity of the ilv2 protein by up to 7-fold (from 6.8 +/- 0.7 to 49.0 +/- 1.8 U/mg) and confers upon it sensitivity to inhibition by valine (Ki = 0.16 +/- 0.02 mM). Valine inhibition is partially reversed by ATP. The reconstitution is favored by high concentrations of potassium phosphate ( approximately 1 M) and at neutral pH. Under optimal conditions for reconstitution, a dissociation constant for the subunits of 70 +/- 7 nM was determined. Valine inhibition is partial, resulting in a specific activity that is similar to that of the ilv2 protein alone. However, measurements of the Km for substrate rule out the possibility that valine inhibition is accomplished by dissociation of the subunits.