The papillomavirus E2 protein is required for viral transcriptional regulation and replication. The E2 protein has a modular structure with two highly conserved domains, a sequence-specific DNA-binding and dimerization domain and a conserved N-terminus which is important for transcriptional transactivation, replication, and interaction with the E1 protein to determine which specific amino acids or regions in the N-terminus were important for the replication or transactivation functions. Single amino acid substitutions were created at highly conserved, highly charged amino acids in the HPV 11 E2 N-terminus. Each amino acid was mutated to a nonpolar alanine residue or a similarly charged amino acid. The mutated E2 proteins were analyzed for their abilities to support transcriptional transactivation and transient DNA replication and to enhance binding of E1 to the origin of replication. Single amino acid substitutions were identified which were defective for either the replication or transactivation functions, which demonstrated that the replication and transactivation functions within the N-terminus are separable. In several cases different amino acid substitutions at the same site had variable effects on transcription or replication, highlighting the importance of hydrophobic interactions or side chain structure at each site. The replication function appeared to correlate with the ability of E2 to enhance binding of E1 to the origin of replication though these studies also suggest that other functions performed by the E2 protein may be important for replication.