Nitrile hydratase (N-Hase) is an important industrial enzyme used for acrylamide production from acrylonitrile. The deactivation kinetics of NHases in free resting cells of Rhodococcus sp. was presented based on a bi-steady state assumption. Effects of hydration temperature, product concentration and substrate concentration on NHase deactivation were investigated experimentally and correlated with a first order deactivation kinetics. The results showed that the hydration temperature and product concentration were major factors governing the deactivation of NHases under substrate-feeding conditions. When acrylamide concentration was higher than 250 g.L-1, the deactivation of NHases became serious and the bi-steady state assumption was not applicable. When the hydration temperature was controlled at a relatively higher level such as 28 degrees C, the total deactivation rate constant was about 2.8-fold of that at 20 degrees C.