IR4, an early regulatory protein of equine herpesvirus 1 (EHV-1), is not a DNA-binding protein, but interacts with the sole immediate-early protein (IEP) to increase both IEP site-specific DNA-binding and IEP-mediated trans-activation of EHV-1 promoters. To investigate the biological properties of IR4 and ascertain whether this regulatory protein is essential for virus growth, bacterial artificial chromosome methods were employed to generate an IR4-null EHV-1. The IR4 gene was dispensable for EHV-1 growth in non-immortalized equine NBL-6 cells, but virus replication was delayed and was reduced by greater than 10-fold. In addition, replication of the IR4 mutant was abrogated in all other cell types tested, including equine ETCC tumor cells and cells of mouse, rabbit, monkey, and human origin. Further, in contrast to the highly pathogenic parent virus, the IR4 deletion mutant failed to cause disease in the CBA mouse as judged by assessing body weight and clinical signs and was unable to replicate in the murine lung. To define the nature of the block in the replication of the IR4-null virus, molecular analyses were carried out in RK-13 rabbits' cells infected with the IR4-deleted virus and revealed that: 1) the synthesis of the sole IEP was not inhibited; 2) the synthesis of early viral proteins examined was either not affected or was delayed to late times; 3) viral DNA replication was inhibited by more than 99.9%; and 4) synthesis of essential late proteins such as glycoprotein D and glycoprotein K was prevented. These findings indicate that the IR4 protein is required for EHV-1 DNA replication in non-permissive cells, and, like its homologues in other alphaherpesviruses, contributes a function required for virus replication in a variety of cell types.