The UL100 gene of human cytomegalovirus (HCMV) has been shown to encode an envelope glycoprotein that might play an important role in HCMV infection. Using the HCMV UL100 gene as a probe in low stringency hybridization studies, we were able to identify the putative UL100 homologous gene of murine cytomegalovirus (MCMV), strain Smith. The MCMV gene was sequenced and localized to the left end of the EcoRI fragment H on the MCMV physical map. This genomic location is similar to that found for the HCMV UL100 gene. The MCMV UL100 mRNA is 1.6 kb in size and is expressed exclusively in the late stages of infection. The 5' and 3' boundaries of the transcript were determined. The open reading frame (ORF) of the UL100 gene could encode a protein of 371 amino acid residues with a calculated molecular mass of 42 kDa. Computer analysis of the deduced amino acid sequence of this gene predicted the presence of eight transmembrane domains and four N-linked glycosylation sites in the protein. Sequence comparison revealed that this putative protein shares similarity with the predicted UL100 homologs of several other herpesviruses, and is most similar to the HCMV UL100 protein (47% identity).