Adeno-associated virus (AAV) uses three promoters, p5, p19, and p40, to regulate viral gene expression. The p5 and p19 promoters direct the synthesis of the viral regulatory proteins, Rep78 and -68 and Rep52 and -40, respectively. The p5 Rep proteins bind a linear 22-bp sequence, the Rep binding element (RBE), that is within both the terminal repeat (TR) and the p5 promoter. In the absence of helper virus, all four Rep proteins have been shown to reduce transcription from the viral p5 and p19 promoters. In this report, we focus on the roles of these proteins and the RBEs in controlling transcription during a productive infection, that is, in the presence of adenovirus. We find that in the presence of adenovirus, the p5 RBE represses p5 transcription while the RBE in the TR activates p5. However, both the TR RBE and the p5 RBE transactivate the p19 and p40 promoters. The fact that the p5 RBE-Rep complex can transactivate p19 and p40 while repressing p5 suggests that Rep78/68 is both a repressor and a transactivator. Rep repression of p5 is specific for the p5 RBE, as other p5 promoter elements do not support this activity. We also demonstrate that in the presence of adenovirus, the p19 Rep proteins, which do not bind to the RBE, can eliminate repression of the p5 promoter by Rep78 and Rep68. This may occur by the association of Rep52 with Rep78 or Rep68 to produce a Rep78/68-Rep52 protein complex which can be detected in vivo by immunoprecipitation. Finally, two Rep mutants that were deficient in RBE binding and transactivation but positive for p5 repression were identified. These mutants may define interaction domains involved in making contacts with other proteins that facilitate repression. These observations suggest a mechanism for controlling the p5 and p19 mRNA levels during a productive AAV infection.