Cryoelectron microscopy images of HPV16 pseudovirions (PsV) depict that each pentamer of L1 can be occluded with a monomer of L2. Further research suggests that an N-terminal external loop of L2 exists, which is the target of neutralizing and cross-neutralizing antibodies. Here we show that N-terminal L2 cysteine residues, Cys22 and Cys28, have overlapping and independent structural roles, which affect both early- and late-stage assembly events. Substitution of either cysteine residue enhances infectivity markedly in comparison to wild-type HPV16. However, only Cys22Ser 20-day virions become nearly as stable as wild type. In addition, Cys22Ser, and Cys22,28Ser 20-day virions have lost their susceptibility to neutralization by anti-L2 antibodies, whereas Cys28Ser 20-day virions remain partially susceptible. These results suggest that Cys28 is necessary for late-stage stabilization of capsids, while Cys22 is necessary for proper display of L2 neutralizing epitopes.