Although the guinea pig cytomegalovirus (GPCMV) model is well suited to the study of vaccines for prevention of congenital CMV infection, there has been limited molecular characterization of GPCMV glycoproteins. Since the in vivo co-expression of the human cytomegalovirus (HCMV) glycoprotein H (gH, gpUL75) with glycoprotein L (gL, gpUL115) may have relevance to CMV vaccine studies, these experiments were undertaken to test whether the GPCMV encodes a gL homolog. Sequencing of the EcoR I 'G' fragment of the GPCMV genome identified an open reading frame (ORF) of 774 nucleotides capable of encoding a protein of 258 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gL coding sequences of other betaherpesviruses. Sequence analysis also identified an ORF with identity to the HCMV uracil DNA glycosylase (UDG, UL114 gene). The GPCMV gL ORF encodes 6 cysteine residues, contains 3 potential N-linked glycosylation sites, and has a predicted M(r) of 29.7 kDa. Northern blot studies identified an abundant 2.7 kb 'early' transcript from infected cells, the putative gL message. In vitro translation of gL mRNA in reticulocyte lysate resulted in synthesis of 30 kDa polypeptide. A polyclonal antiserum was raised against a gL/glutathione-S-transferase fusion protein generated in E. coli using the pGEX expression system. This antibody identified a 40-kDa virion-associated protein, the putative GPCMV gL, in immunoblot assays.