Transcription initiation by mammalian RNA polymerase II is effected by multiple common factors interacting through minimal promoter elements and regulated by gene-specific factors interacting with distal control elements. Minimal promoter elements that can function independently or together, depending on the specific promoter, include the upstream TATA box and a pyrimidine-rich initiator (Inr) overlapping the transcription start site. The binding of TFIID to the TATA element promotes the assembly of other factors into a preinitiation complex but factors which function at the Inr have not been defined. We show here that a novel factor (TFII-I) binds specifically to Inr elements, supports basal transcription from the adenovirus major late promoter and is immunologically related to the helix-loop-helix activator USF. We further show that TFII-I also binds to the upstream high-affinity USF site (E box), that USF also binds to the Inr, and that TFII-I and USF interact cooperatively at both Inr and E box sites. Thus, TFII-I represents a novel type of transcription initiation factor whose interactions at multiple promoter elements may aid novel communication mechanisms between upstream regulatory factors and the general transcriptional machinery.