Transcription directed by the BPV-1 long control region (LCR) is conditional upon activation by the virally encoded E2 protein. Within the 1.0 kb LCR there are five separate regions, A to E, that contain E2 responsive enhancers. The smallest functional region, A, is only 38 bp and contains two copies of the consensus sequence ACC(N)6GGT that is known to function as an E2 binding site in vitro. We show that a pair of these constitutes a minimal functional E2 responsive enhancer element but that the strength of enhancer activity is dramatically reduced both by increasing the spacing between them and by removing the dual elements from the proximity of other key promoter elements. Furthermore, pairs of dual elements activated transcription to varying levels depending upon their spatial arrangement and promoter proximity. We have also identified a low level constitutive enhancer in the D region which lacks an E2 consensus binding site but which can be activated by E2. We show that the activation potential of this constitutive enhancer is increased by association with a single E2 binding site suggesting some cooperation/interaction between viral and cellular enhancer proteins.