A strain of Rhodococcus equi SHB-121 forming 3-cyanopyridine hydratase was screened from nitrile-polluted soil. The optimum conditions for the formation of 3-cyanopyridine hydratase by the strain SHB-121 have been studied. Under the optimum conditions, the specific activity of the enzyme reached 5.32 U/mg of dry cell, 95 times higher than that cultured in screening medium. In addition, the activity of coexistent amidase was very low. 3-Cyanopyridine hydratase was purified from methylacry-lamide-induced cells of Rh, equi SHB-121 by procedures including ultrasonic oscillation, ammonium sulfate precipitation, and column chromatographies on DEAE-cellulose DE52, hydroxyapatite, and Sephadex G-25. The overall purification was 31-fold. The molecular weight of the enzyme was about 30 LDA by SDS-PAGE. The pi value was 4.1. The transition temperature and pH were 7.0 degrees C and 6.0, respectively, resulting from the differential spectra. The optimum pH and temperature for the enzyme reaction were 8.0 and 30 degrees C. The enzyme activity was strongly inhibited by Ag+, Hg2+, Cu2+, and NH4+, whereas it was enhanced by Fe3+ slightly. The enzyme catalyzed the hydration of 3-cyanopyridine to nicotinamide, and its Km value was 0.1 mol/L. Uncompetitive inhibitor sodium cyanide has a K-i value of 5 mmol/L.