Encapsidation of papillomavirus DNA involves DNA-protein and protein-protein interactions. We sought to define the role of each human papillomavirus (HPV) capsid protein in HPV DNA encapsidation. HPV16 major (L1) and minor (L2) capsid proteins purified from recombinant vaccinia virus-infected cells were compared for their ability to bind nucleic acids. L2 protein, but not L1 protein, could bind HPV DNA. To map the DNA-binding region of L2, a series of truncated or point-mutated L2 protein open reading frames were used to show that only the N terminal of L2 was required for L2-DNA binding. This interaction depends critically on charged amino acids (Lys or Arg) in the first 12 amino acids of the N terminal of the protein. Several techniques were used to show that L2 interaction with DNA did not require specific DNA sequences. We propose that HPV L2 protein may play a major role in papillomavirus capsid assembly by introducing HPV DNA to the virus particles formed by the self assembly of the L1 major structural protein.