Human papillomavirus type 16 (HPV-16) DNA replicates episomally and requires two virally expressed proteins, E1 and E2. The E1 protein has both helicase and ATPase activities and is absolutely required for viral DNA replication. The E2 protein is a potent transcriptional activator and greatly increases viral DNA replication by colocalizing E1 to the origin of replication. Recently, we characterized a region of the E2 protein essential for the binding to E1. In this study we have analysed in further detail the nature of the association between E1 and E2. Using an extensive set of E2 mutant proteins we have identified two widely separate regions of the E2 protein which are essential for binding to E1. Interestingly, two E2 mutants which fail to bind E1 also fail to activate gene expression, indicating the existence of multifunctional domains on the E2 protein. In addition, cotransfection of E1 with E2 significantly increases E2 transcriptional activity on an heterologous promoter.