Autographa californica nuclear polyhedrosis virus (AcMNPV) contains five homologous regions (hr1 through hr5) interspersed throughout its genome. Analysis of plasmid transfections indicates that the hrs function as transcriptional enhancers and possible origins of viral DNA replication. The role of these repetitive elements in regulating expression from the AcMNPV genome was examined by constructing a series of recombinant viruses that tested the effect of hr5 on expression of the adjacent p35 gene (p35). When embedded within the viral genome, hr5 stimulated transcription from the early p35 promoter in a position- and orientation-independent manner. Moreover, hr5 and the upstream activating region of p35 were functionally interchangeable. A 28-bp imperfect palindrome, repeated six times within hr5, was the minimal sequence required for p35 promoter activation. hr5 also stimulated another early AcMNPV promoter but not a late promoter or a host-derived promoter, suggesting that enhancement is promoter specific during infection. To investigate its role during AcMNPV replication, hr5 was deleted from its normal position within the viral genome. The resulting hr5 mutants exhibited no apparent defects in replication, as judged by production of budded virus and levels of very late gene expression, even though steady-state levels of p35 RNA were reduced. These results indicated for the first time that hr5 functions as a transcriptional enhancer within the viral genome. However, the element is not required for AcMNPV replication in cultured cells. Thus, loss of one of five possible origins of DNA replication is not deleterious to viral growth. Since p26 was removed from the hr5 deletion mutants, this gene is also nonessential for viral replication.