This work aims to produce alpha-ketoisocaproate (KIC) from L-leucine via the free-whole-cell biotransformation of Rhodococcus opacus DSM 43250. The effects of temperature, pH, substrate concentration, cell concentration, and rotating speed on KIC production were examined. Furthermore, the biotransformation conditions were optimized with response surface methodology (RSM). The optimal biotransformation conditions were as follows: temperature 43.7 degrees C, pH 8.4, L-leucine concentration 5.1 g/L, cell concentration 30.4 g/L, and rotating speed 170 rpm. The maximal KIC production predicted by RSM model reached 1275 mg/L at the optimal conditions, and in the validated experiments, the maximal KIC production reached 1264 mg/L, which was very close to the predicted KIC production. The current work provides an alternative for the production of KIC and the results obtained here are useful for the biotransformatic KIC production on a large scale. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.