Polymerase II transcription of a human gene for the small nuclear RNA U2 is dependent on two different promoter elements: a TATA-equivalent proximal sequence element and a distal enhancer element, which has been shown to contain Sp1- and octamer-binding sites. We have investigated the functional interplay between these transcription factor-binding sites of the enhancer, following transfection of U2 maxigene constructions into HeLa cells. There is a functional non-additive co-operation between the octamer-binding factor and Sp1, which is not dependent on the evolutionally conserved steric arrangement of these binding sites. We demonstrate that the conserved Sp1-binding site of the U2 enhancer can be fully substituted by a nuclear factor I (NFI) binding site, and that the octamer-binding factor functions in stimulating transcription in conjunction with either Sp1 or NFI. Since the octamer-binding factor is most probably the same protein as nuclear factor III (NFIII), the results imply that the NFI NFIII complex, involved in adenovirus DNA replication, also can function as an efficient activator of transcription.