The Autographa californica multinucleocapsid polyhedrosis virus homologous region sequence hr1 enhances transcription from the viral polyhedrin promoter in Spodoptera frugiperda insect cells and independently functions as an origin of replication (ori) sequence. The binding of the host nuclear protein, hr1-binding protein (hr1-BP), is crucial for the enhancer activity (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737-747 and Habib, S., and Hasnain, S. E. (1996) J. Biol. Chem. 271, 28250-28258). We demonstrate that hr1 can also enhance transcription from non-baculoviral promoters like cytomegalovirus and hsp70 in mammalian cells but does not support ori activity in these cells. Unlike insect cells, hr1 can also function in mammalian cells as an enhancer when present in trans. hr1 DNA sequence binds with high affinity and specificity to nuclear factors in the mammalian cells. The insect hr1-BP- and the hr1-BP-like proteins from mammalian cells (mhr1-BP) have different properties with respect to ion requirements, DNA groove binding, and molecular size. When mammalian cells are infected with a recombinant baculovirus containing two promoters, the baculovirus polyhedrin and Drosophila hsp70 gene promoter, the hsp70 gene promoter alone is active in these cells, and this activity is further enhanced by the presence of an additional hr1 in the recombinant virus. hr1 may thus also have a role in baculovirus-mediated gene delivery in mammalian cells.