BACKGROUND: The chemo-selective biocatalytic hydrolysis of nitriles presents a valuable alternative to chemical hydrolysis, a procedure in which harsh conditions are applied. In this study, Rhodococcus ruber CGMCC3090 is used for 5-cyanovaleramide (5-CVAM) production by means of adiponitrile (ADN) hydration. Several parameters that affect the biocatalyzation process are investigated. RESULTS: The effective production of 5-CVAM from ADN with good regioselectivity was successfully achieved using the resting cells of Rhodococcus ruber CGMCC3090. The reaction parameters for 5-CVAM production were investigated during the experiment, and it was found that resting cells were effective in converting ADN at high concentrations (up to 2.0 mol L-1) and the hydration reaction was slightly inhibited by different concentrations of 5-CVAM. After 100 min of incubation, more than 99.2% of the added adiponitrile was converted to 5-CVAM when the cell concentration was 1.05 g dry cell weight (DCW) L-1. CONCLUSION: The study provides a facile and feasible way of achieving high 5-CVAM production levels while maintaining high purity of the product obtained. Copyright (c) 2012 Society of Chemical Industry