Technical Abstract: Marek’s disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens, called Marek’s disease (MD). In the unique long region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits of ribonucleotide reductase (RR) enzyme, named RR1 and RR2, respectively. MDV RR is distinguishable from that present from chicken and duck cells by monoclonal antibody T81. Using recombinant DNA technology we have generated a mutant MDV (Md5'RR1) in which RR1 was deleted. PCR amplification of RR gene in rMd5'RR1-infected DEF confirmed the deletion of 2.4 Kb RR1 with a resultant amplicon of a 640-bp fragment. Restriction enzyme digests with SalI confirmed UL39 deletion and absence of rearrangement. The biological characteristics of Md5'RR1 virus was studied in vitro and in vivo. The Md5'RR1 replicated in duck embryonic fibroblasts but significantly slower than that of parental Md5BAC, suggesting that RR is required but not essential for replication in fibroblasts. In vivo studies, however, showed that RR1 deletion virus was impaired for its ability to replicate in chickens. Inoculation of SPF chickens with Md5'RR1 showed the mutant virus is non-pathogenic and does not induce MD in birds. A revertant virus, Md5'RR1/R, was generated with the restored phenotype of the parental Md5-BAC in vivo, indicating that RR is essential for replication of the virus in chickens. Protection studies in SPF chickens indicated that Md5'RR1 virus is not a candidate vaccine for MD.