By in vitro analysis, the major late promoter (MLP) of adenovirus has been shown to be a simple promoter requiring two elements for efficient transcription: a minimal promoter element (MPE), where the general transcription factor-polymerase II complex binds, and a single functional upstream promoter element (UPE) which interacts with USF. Two hundred bases upstream of the MLP cap site and divergently oriented is the IVa2 promoter. This promoter has its own MPE but shares the MLP UPE, suggesting the possibility that these promoters are coordinately regulated. To determine mechanistically how this might function, we replaced the weak IVa2 minimal promoter with a strong MPE (from the viral E1A gene) and observed mutual inhibition of both promoters and unstable transcription factor binding. Only by duplication of the UPE could this inhibition be relieved. When tested independently, both promoters were shown to require the USF site for maximal activity. These results are compatible with a model in which USF can stably interact with only one transcription complex at a time. When two divergently oriented general transcription complexes compete efficiently for binding of USF, transcription is reduced to the same levels as if the USF site were absent.