Prophylactic vaccines against Human Papillomavirus (HPV) based on Virus-Like Particles (VLPs) induce type-specific neutralizing antibodies. The major capsid protein L1 is highly conserved between HPV types, with a small number of hypervariable residues positioned in surface-exposed loops. To investigate the importance of these residues for the immunogenicity of HPV 16 and the L1 interaction with minor capsid protein L2, ten surface-exposed amino acid residues in 4 hypervariable loops of L1 were mutated. VLPs containing mutated or wild type L1 with or without L2 were produced in 293TT cells using pseudovirion expression vectors. The mutations reduced, but did not eliminate, the ability to bind neutralizing monoclonal antibodies and to induce neutralizing antibodies and cytotoxic T- lymphocyte responses. Cross-neutralization was not detected. The mutations in the L1 loops caused a strikingly decreased incorporation of L2 protein and a complete inability to encapsidate pseudogenomes, suggesting that intact L1 loops are important for the interaction of L1 with L2 in the HPV particle.