We have identified a gene, lpt-3, that is required for the addition of phosphoethanolamine to the 3-position (PEtn-3) on the beta-chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn-3 substituent is characteristic of the LPS of a majority ( approximately 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface-accessible epitope. All strains of Nm that have PEtn-3 possess the lpt-3 gene. In some lpt-3-containing strains, the 3-position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt-3 resulted in loss of PEtn-3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt-3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS-based vaccine to protect against invasive meningococcal disease.