Conditions for renaturation and purification of recombinant D-2-hydroxyisocaproate dehydrogenase (r-DHicDH) produced in E. coli in the form of inclusion bodies were investigated. Urea (8 M) was found to extract the enzyme from the pellet fraction efficiently. Renaturation by dialysis against 20 mM sodium phosphate buffer, pH 7.0, a protein concentration of 2 mg ml(-1), and a temperature of 4-8 degrees C was found to be optimal and even at a protein concentration of 10 mg ml(-1) 85% of enzyme activity could be recovered. Protein was further purified by ion-exchange chromatography on a Mono Q column. An enzyme preparation of greater than 95% purity was obtained with an overall yield of about 50%. r-DHicDH shows identical properties within experimental error with respect to molecular weight, temperature dependence of enzyme activity, pH optimum of the enzyme reaction, and K-M values for 2-ketoisocaproate and NADH as the native enzyme from Lactobacillus casei.