ABSTRACT We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, P _F , which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 Delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for Delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the −35 region of the promoter. Introduction of a σ ⁷⁰ −35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by Delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor σ ⁷⁰ contacts and helps to recruit RNA polymerase holoenzyme.