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AIMS The efficiency of acrylamide production was examined with immobilized cells of Rhodococcus rhodochrous (RS-6) containing NHase. METHODS AND RESULTS Different entrapment matrices such as agar, alginate and polyacrylamide were used. Various immobilization parameters like agar concentration, cell concentration and reaction conditions affecting the bioconversion process using suitable matrices were determined. The cells immobilized with agar matrix were found to be most effective for acrylonitrile conversion. The bioconversion was more efficient in beads prepared with 2% agar and 5% (v/v) cell concentration. The entire conversion of acrylonitrile to acrylamide with agar entrapped cells was achieved in 120 min at 15°C. The agar entrapped R. rhodochrous (RS-6) cells exhibited 8% (w/v) tolerance to acrylonitrile and 35% tolerance to acrylamide. The immobilized cells also retained 50% of its conversion ability up to seven cycles. The laboratory scale (1 L) production resulted in 466 g L-1 accumulation of acrylamide in 16 h. CONCLUSIONS The cells immobilized in agar showed better stability and biocatalytic properties and increased reusability potential. SIGNIFICANCE AND IMPACT OF THE STUDY The agar immobilized Rhodococcus rhodochrous (RS-6) cells showed enhanced tolerance for both the substrate and product and is economical for the large scale production of acrylamide.