The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. In order to study the biological function of LORF11 we deleted it from the MDV cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method. The recombinant cosmid, A6ΔLORF11, was transfected into duck embryo fibroblasts (DEF) in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. Recombinant rMd5ΔLORF11 plaques were evident at 12–13 days after transfection. Polymerase chain reaction amplification of DEF cells infected with rMd5ΔLORF11 viruses confirmed the deletion of a 2.57-kb fragment resulting in a 296-bp fragment. Three rMd5ΔLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5ΔLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5ΔLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100× lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5ΔLORF11-infected chickens. Three independent deletion mutants were tested and showed the same phenotypes, so it is unlikely that the observed phenotype is because of any random mutation in the genome. Therefore the LORF11 gene of MDV is essential for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus.