Heterogenous expression of active nitrile hydratase (NHase) was focused for its great potential in genetically evolution of the operational stability of NHase. Two recombinant Escherichia coli strains, E. coli JM105 (pUC18-NHBAX) and E. coli BL21 (DE3) (pET32a-NHBAX), were first constructed and used for heterogenous expression of a NHase gene cloned from Nocardia YS-2002. It was found that the alpha subunit of NHase can not be effectively expressed in both recombinant E. coli, which results in as low as 0.04U/mg (dry cell weight) activity of NHase in E. coli BL21 (DE3) (pET32a-NHBAX), and no activity in JM105 (pUC18-NHBAX) at all. Therefore, the recombinant and active expression of NHase in Pichia pastoris was especially interested. A new plasmid, pPIC3.5k-NHBAX, was constructed by insertion of the NHase gene, and then successfully transformed into the cell of Pichia pastoris GS115 by electroporation. A novel superior strain, P. pastoris NH4, was selected from 6 target-clones and used for optimization of cell culture and NHase expression. The final activity of NHase expressed in P. pastoris NH4 under optimal conditions is 0.52U/mg (dry cell weight), which is 13-fold higher than that in recombinant E. coli. However, the activity of NHase expressed in recombinant P. pastoris NH4 can not be stably maintained longer than 6 h.