The bacteriophage T4 AsiA protein is a bifunctional regulator that inhibits transcription from the major class of bacterial promoters and also serves as an essential co-activator of transcription from T4 middle promoters. AsiA binds the primary s factor in Escherichia coli, sigma(70), and modifies the promoter recognition properties of the sigma(70)-containing RNA polymerase(RNAP) holoenzyme. In its role as co-activator, AsiA directs RNAP to T4 middle promoters in the presence of the T4-encoded activator MotA. According to the current model for T4 middle promoter activation, AsiA plays an indirect role in stabilizing the activation complex by facilitating interaction between DNA-bound MotA and sigma(70). Here we show that AsiA also plays a direct role in T4 middle promoter activation by contacting the MotA activation domain. Furthermore,we show that interaction between AsiA and the beta-flap domain of RNAP is important for co-activation. Based on our findings, we propose a revised model for T4 middle promoter activation, with AsiA organizing the activation complex via three distinct protein-protein interactions.