Abstract We have identified three Marek's disease virus (MDV) open reading frames (ORFs) within the BamHI D fragment of MDV genome. The predicted polypeptides are homologous to UL1 (glycoprotein L, gL), UL2 (uracil-DNA glycosylase), and UL3 (nuclear localizing phosphoprotein) of herpes simplex virus type 1 (HSV-1). Comparison of the deduced amino acid sequences of these three ORFs with HSV-1 counterpads revealed overall identities of 18, 43, and 49%, respectively. In spite of the low overall amino acid identity with HSV-1 gL, the first open reading frame was identified as a gL homolog of HSV-1 based not only on the gene arrangement but also on a limited amino acid conservation among gL homologs of alpha-herpesviruses. To characterize the expression of the MDV gL gene, an antiserum to a hydrophilic region of the gene expressed in a bacterial expression vector was produced. Immunoprecipitation with this antiserum revealed a 25,000-Da polypeptide in MDV-infected cells. Furthermore, the 25,000-Da polypeptide migrated as a 18,000-Da polypeptide following PNGase F treatment. This result is consistent with the predicted molecular weight of MDV gL, considering the two potential Nglycosylation sites and the predicted N-terminal signal sequence. A recombinant fowlpox virus expressing the MDV gL gene was generated to characterize this glycoprotein. Unlike gL in MDV-infected cells, gL expressed by recFPV-gL was highly sensitive to Endo H, indicating that it was probably retained in the endoplasmic reticulum and was not properly processed to a mature form. Therefore, similar to HSV-1, coexpression and complex formation of MDV gL and gH may be required for proper processing and transport of gL to the cell surface.