We have investigated bacterial factors required for the entry of Neisseria meningitidis serogroup B into mucosal cells using a novel in vitro infection model of primary cultures of human nasopharyngeal epithelium. An invasive meningococcal phenotype was obtained after several cycles of selection for intracellular bacteria with gentamicin. Invasive bacteria differed from those in the initial inoculum in that they lacked a capsule and pili, exhibited a nonsialylated low-molecular-weight type of lipopolysaccharide (LPS), and produced a new 28-kDa opacity outer membrane protein. LPS revertants of the selected meningococci expressed a nonsialylated L3,(7,9) type of LPS and were also invasive, while after LPS sialylation bacterial entry was inhibited. Variants lacking the 28-kDa opacity protein were poorly invasive. Coexpression of the outer membrane protein Opc and the 28-kDa opacity protein strongly inhibited microbial invasion into the primary cultured nasopharyngeal cells. Conversely, meningococcal internalization by cells of various epithelia] cell lines was correlated with the expression of Opc rather than the 28-kDa opacity protein. Our data indicate that a concurrent phase switching of multiple phase-variable bacterial surface components may be a prerequisite for meningococcal invasion into nasopharyngeal epithelium and that meningococcal class 5 proteins (Opa and Opc) may promote tissue tropism.