The interactions of Neisseria meningitidis with cells of the meninges are critical to progression of the acute, compartmentalized intracranial inflammatory response that is characteristic of meningococcal meningitis. An important virulence mechanism of the bacteria is the ability to shed outer membrane (OM) blebs containing lipopolysaccharide (LPS), which has been assumed to be the major pro-inflammatory molecule produced during meningitis. Comparison of cytokine induction by human meningeal cells following infection with wild-type meningococci, LPS-deficient meningococci or after treatment with OM isolated from both organisms, demonstrated the involvement of non-LPS bacterial components in cell activation. Significantly, recognition of LPS-replete OM did not depend on host cell expression of Toll-like receptor (TLR)4, the accessory protein MD-2 or CD14, or the recruitment of LPS-accessory surface proteins heat shock protein (HSP)70, HSP90α, chemokine receptor CXCR4 and growth differentiation factor (GDF)5. In addition, recognition of LPS-deficient OM was not associated with the expression of TLR2 or any of these other molecules. These data suggest that during meningococcal meningitis innate recognition of both LPS and non-LPS modulins is dependent on the expression of as yet uncharacterized pattern recognition receptors on cells of the meninges. Moreover, the biological consequences of cellular activation by non-LPS modulins suggest that clinical intervention strategies based solely on abrogating the effects of LPS are likely to be only partially effective.