Infection of shrimps with white spot syndrome virus (WSSV) characterizes in fast virus replication. Therefore, the viral enzymes involved in DNA synthesis and metabolism must be very important. Among these enzymes, WSSV-encoded ribonucleotide reductase (RR) catalyzes the conversion of ribonucleoside diphosphates (NDP) to the corresponding deoxyribonucleoside diphosphates (dNDP), and thus plays a major role in DNA synthesis regulation. Moreover, previous studies show that the mRNA expression of both RR subunits increases concomitantly with the advancement of WSSV infection. A baculovirus/insect system was used to express the two recombinant subunits, RR1 and RR2. The SDS-PAGE electrophoresis showed high expression level of both recombinant subunits. The co-immunoprecipitation assay demonstrated that recombinant WSSV-RR1 and –RR2 reassembled in vitro. The DNA polymerase coupled RR activity assay showed a marked increase in RR activity when cell extracts containing recombinant RR1 and RR2, which are both required for activity, were combined. These experiment results demonstrated that recombinant WSSV-RR1 and –RR2 reassembled in vitro and formed active holoenzyme to catalyze the reaction. We found that EDTA and hydroxyurea inhibited rWSSV-RR activity. This suggests that metal ions and free radicals are required for its activity which is similar to the cases on eukaryotic cellular RRs. The reduction of cytidine 5’-diphophate (CDP) catalyzed by the recombinant WSSV RR was accelerated by ATP, a general activator of RR, in an optimal range of concentration. However, WSSV RR is different from the insect cellular RRs by its insensitivity to allosteric inhibition by dTTP and dATP. It is suggested that the tolerance of rWSSV-RR and cellular RR in feedback inhibition is different. RR activity increased in the gills and heart of experimentally infected shrimp as infection advanced. The increase is much higher in the gills than in the heart. Western blot analysis using specific polyclonal antibody raised against recombinant protein shows a concomitant increase in RR protein expression in WSSV-infected shrimp stomach tissues as the infection progresses. These results suggest that WSSV RR is functionally involved during WSSV infection.