3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K-m values for 3-isopropylmalate and NAD(+) were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degreesC. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.