A new gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) has been identified that encodes a highly basic 8.9K protein. The gene called p8.9 is expressed as a 0.5 kb transcript by 1 h post-infection and initiates at an early gene motif. The promoter of the 0.5 kb transcript has two upstream elements, repeats I and II, which are similar to motifs previously characterized in the OpMNPV IE-2 gene and the Autographa californica nuclear polyhedrosis virus IE-N and PE38 genes. A second p8.9 transcript expressed from 8 to 72 h post-infection was shown to initiate 634 bp upstream from the early gene motif in a region that has no similarity to any previously described baculovirus promoter or initiation site. Transient assays utilizing a reporter gene have shown that the p8.9 early promoter is active in a Lymantria dispar (LD652Y) and Spodoptera frugipeda (Sf9) cell lines in the absence of other viral factors. In addition, it was also demonstrated that the p8.9 promoter is trans-activated by the OpMNPV IE-2 and p34 genes, but not by IE-1.