A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l(-1)) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l(-1), 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application.