Activity of the adeno-associated virus P5 transcriptional control region was found to be induced by adenovirus E1A gene products. A pair of adjacent sequence elements was found to mediate both basal and E1A-induced P5 activity. The first element is a binding site for the major late transcription factor (MLTF), a factor first identified on the basis of its binding to a specific sequence within the adenovirus major late promoter. The second element is a tandemly repeated 10-base-pair sequence whose relationship to previously described binding sites is unclear. Each element individually conferred E1A responsiveness on a heterologous promoter, and deletion analysis demonstrated that each contributed to the level of P5 activity in the presence of E1A products both in transfection- and infection-based assays. Although deletion of the MLTF binding site led to reduced P5 transcriptional activity in the presence of E1A proteins, the deletion generated enhanced P5 basal activity in the absence of the transcriptional activator. The negative effect of the MLTF binding site in the absence of activator and its positive effect in the presence of activator combine to enhance the magnitude of the response by the P5 control region to E1A gene products.