The contributions of the glycoproteins gI (ORF67) and gE (ORF68) to varicella-zoster virus (VZV) replication were investigated in deletion mutants made by using cosmids with VZV DNA derived from the Oka strain. These experiments demonstrated that gI was not required for VZV replication in vitro but gE appeared to be. Although VZV gI was not required, its deletion or mutation resulted in a significant decrease in infectious virus yields, and it disrupted syncytial formation and altered the conformation and distribution of gE in infected cells. Normal cell-to-cell spread and replication kinetics were restored when gI was expressed from a non-native locus in the VZV genome. The expression of intact gI, the ORF67 gene product, is required for efficient VZV replication.