Pseudorabies virus (PRV) early protein O (EPO) is the homolog of varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein and herpes simplex virus type 1 (HSV-1) ICPO. A PRV EPO deletion mutant grows poorly in cell culture, suggesting that EPO plays a critical role in the viral replicative cycle. In this study, we have shown that the growth defect of an EPO deletion mutant was complemented in Vero cells expressing VZV ORF61 protein or HSV-1 ICPO. In transient expression assays PRV EPO, like VZV ORF61 protein and HSV-1 ICPO, transactivates a variety of promoters from PRV, VZV, HSV, and unrelated viruses. These data indicate that PRV EPO is functionally homologous to VZV ORF61 protein and HSV-1 ICPO.