The five regions of homologous DNA which are interspersed in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus increased the expression of a delayed-early gene of this virus. Although this activity was first observed as a 10-fold trans effect, the homologous region 5 (hr5) enhanced the expression of linked genes 1,000-fold. The hr5 enhancer also exhibited the other characteristics associated with viral enhancer elements, including orientation independence and the abilities to function at a distance from the linked promoter, to regulate heterologous promoters, and to increase the number of RNA polymerase molecules transcribing the linked genes. The expression of the immediate-early regulatory gene was not enhanced by cis-linked hr5, although the enhancer function may require the immediate-early regulatory gene product. The hr5 enhancer was relatively insensitive to competition by an excess of enhancer molecules. The nucleotide sequence of hr5 revealed two different conserved repeats separated by nonhomologous DNA. Deletion analysis of the hr5 enhancer indicated that a 30-base-pair inverted repeat was essential for enhancer function.