Abstract The upstream promoter region of the Autographa californicamulticapsid nuclear polyhedrosis virus (AcMNPV) gp64gene contains five copies of TAAG, the conserved sequence found at the transcriptional initiation sites of almost all baculovirus late genes. In AcMNPV-infected Sf9 cells, late transcription initiation is detected from only two upstream TAAG sites and not from three downstream TAAG sites. To examine several models for preferential TAAG site utilization, we constructed a series of recombinant AcMNPV baculoviruses that contain promoter region sequences from the gp64gene fused to a chloramphenicol acetyl transferasereporter gene. Promoter–reporter constructs were inserted in the polyhedrin locus. To test a scanning model in which TAAG sites are sequentially selected according to their location in the region, we generated recombinant viruses in which the highly transcribed sites were inactivated by point mutations. Transcription from the mutant promoter constructs was compared qualitatively and quantitatively to transcription from the wild-type gp64promoter. Inactivation of the upstream TAAG sites did not result in increased transcription from the downstream TAAG sites, suggesting that immediate context, rather than position, determines promoter utilization. To test this hypothesis, we made a series of minimal promoter constructs containing decreasing quantities of the sequences immediately flanking one of the active gp64TAAG sites. Reporter constructs containing a gp64TAAG site and ≥12 bp of flanking sequence on both sides were transcribed at near wild-type levels. Constructs with less flanking sequence (9 or 6 bp of flanking sequence) were accurately transcribed, but at substantially lower levels, and transcription was not detected from constructs containing only 3 bp of flanking sequence. These results suggest that nucleotides immediately flanking the TAAG site (4–6 bp) are necessary for basal promoter activity while additional flanking sequences (≥12 bp) are required for late promoter activation and regulation. To further examine late promoter selection, we constructed recombinant AcMNPV baculoviruses that contain heterologous late promoters from the gp64gene of the related virus Orgyia pseudotsugataMNPV (OpMNPV). TAAG sites that serve as functional late promoters in OpMNPV were found to mediate transcription initiation at only basal levels in the context of the AcMNPV genome, suggesting that late promoter activation may be virus specific within the family Baculoviridae.