The role of the products of the UL10 and the UL49.5 homologous genes of Marek's disease virus serotype 1 (MDV-1) in virus replication was investigated. Deletion of either open reading frame in an infectious bacterial artificial chromosome clone (BAC20) of MDV-1 resulted in progeny viruses that were unable to spread from cell to cell. After transfection of UL10- or UL49.5-negative BAC20 DNA into chicken or quail cells, only single infected cells were observed by indirect immunofluorescence analysis. In contrast, plaque formation was restored when mutant BAC DNAs were co-transfected with the corresponding expression plasmid encoding either the UL10-encoded gM or the UL49.5 gene product. These data demonstrate that gM and its putative complex partner, the UL49.5 homologous protein, are essential for MDV-1 growth in cultured cells. Thus, MDV-1 represents the first example of a member of the family Herpesviridae for which the highly conserved membrane proteins are indispensable for cell-to-cell spread.