The non-structural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV) functions as a 3C-like proteinase (3CLpro) and plays a pivotal role in gene expression and replication. We have examined the biochemical properties of PRRSV 3CLpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-PRRSV strategies. The 3C-like proteinase (3CLpro) of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and characterized. The optimal temperature and pH for its proteolytic activity were 8°C and 7.5, respectively. Na(+) (1000 mM) and K(+) (500 mM) were not inhibitory to its activity but Cu(2+), Zn(2+), PMSF and EDTA were significantly inhibitory. His(39), Asp(64) and Ser(118) residues were identified to form the catalytic triad of PRRSV 3CLpro by a series of site-directed mutagenesis analysis.