NspA is a highly conserved membrane protein that is reported to elicit protective antibody responses against Neisseria meningitidis serogroups A, B and C in mice (D. Martin, N. Cadieux, J. Hanel, and B. R. Brodeur, J. Exp. Med. 185:1173-1183, 1997). To investigate the vaccine potential of NspA, we produced mouse anti-recombinant NspA (rNspA) antisera, which were used to evaluate the accessibility of NspA epitopes on the surface of different serogroup B strains by an immunofluorescence flow cytometric assay and by susceptibility to antibody-dependent, complement-mediated bacteriolysis. Among 17 genetically diverse strains tested, 11 (65%) were positive for NspA cell surface epitopes and 6 (35%) were negative. All six negative strains also were resistant to bactericidal activity induced by the anti-rNspA antiserum. In contrast, of the 11 NspA surface-positive strains, 8 (73%; P < 0.05) were killed by the antiserum and complement. In infant rats challenged with one of these eight strains, the anti-rNspA antiserum conferred protection against bacteremia, whereas the antiserum failed to protect rats challenged by one of the six NspA cell surface-negative strains. Neither NspA expression nor protein sequence accounted for differences in NspA surface accessibility, since all six negative strains expressed NspA in outer membrane preparations and since their predicted NspA amino acid sequences were 99 to 100% identical to those of three representative positive strains. However, the six NspA cell surface-negative strains produced, on average, larger amounts of group B polysaccharide than did the 11 positive strains (reciprocal geometric mean titers, 676 and 224, respectively; P < 0.05), which suggests that the capsule may limit the accessibility of NspA surface epitopes. Given these strain differences in NspA surface accessibility, an rNspA-based meningococcal B vaccine may have to be supplemented by additional antigens.