In BmN cells infected with the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), IE1, a principal transcriptional activator, localizes to sites of viral DNA replication. IE1 initially displays focal distribution in BmNPV-infected cells prior to DNA synthesis, whereas the protein expressed by transfection with the ie1 gene is distributed throughout the nucleoplasm instead of localized to discrete subnuclear structures. To identify the inducer of focus formation for IE1, we conducted transfection experiments with an IE1-GFP construct and found that cotransfection with genomic DNA fragments bearing the homologous region (hr) sequences caused the formation of IE1-green fluorescent protein (GFP) foci. The transfection of insect cells with a single plasmid containing exclusively the hr3 sequence and the IE1-GFP gene was sufficient to form IE1-GFP foci. These results suggest that hr elements are a primary determinant of the focal distribution of IE1. An analysis of a series of hr3 deletion mutants showed that a single copy of the direct repeat could induce the formation of IE1 foci. Targeted mutagenesis within the hr-binding domain of IE1-GFP caused impairment of the hr-dependent IE1 localization, suggesting that binding of IE1 to the hr elements is essential for the onset of IE1 focus formation. The observation of BmNPV IE1 foci in non-BmNPV-susceptible cells suggests that no species-specific factors are required for hr-dependent IE1 focus formation.