DNA sequence analysis revealed a gene encoding the Marek's disease virus (MDV) DNA polymerase (pol) within the BamHI-E fragment of the long unique region of the virus genome. Identification is based on an extensive amino acid homology between the MDV open reading frame and the DNA pol (UL30) of the herpes simplex virus. We describe here a 3540-base-pair fragment of the MDV DNA encoding 1180 amino acids with a M(r) of 133,920 daltons as the viral DNA pol gene, with the analysis of transcription and translation. In Northern blot hybridization, a transcript of 4.0 kb was detected in GA-MDV-infected duck embryo fibroblast (DEF) cells. An antiserum was generated in rabbit using TryE-pol fusion protein expressed in E. coli. This antiserum specifically immunoprecipitated a protein of 135 kD from lysates of MDV-GA-infected DEF cells. MDV DNA pol showed extensive homology to five distantly related herpesviruses: equine herpesvirus (EHV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV). Comparison of amino acid sequences among the herpesviruses highlights nine highly conserved regions. Three of the conserved regions are in the N-terminus in the 3'-5' exonuclease domains and the remaining six are in the C-terminus in the catalytic domains. The predicted structural characters are in good agreement with the published data on a number of human herpesvirus DNA pol. The identification of MDV DNA pol gene may lead to a better understanding of MDV replication.