Rifampin-resistant (Rifr) Neisseria meningitidis strains are known to have single point mutations in the central conserved regions of the rpoB gene. We have demonstrated two distinct resistance phenotypes in strains with identical mutations in this region, an intermediate level of resistance in Rifr clinical isolates and a high level of resistance in mutants selected in vitro. The possible role of membrane permeability in the latter was investigated by measuring MICs in the presence of Tween 80; values for high-level-resistance mutants were reduced to intermediate levels, whereas those for intermediate-level-resistance strains were unaffected. The highly resistant mutants were also found to have increased resistance to Triton X-100 and gentian violet. Sequencing of the meningococcal mtrR gene and its promoter region (which determine resistance to hydrophobic agents in Neisseria gonorrhoeae) from susceptible or intermediate strains and highly resistant mutants generated from them showed no mutation within this region. Two-dimensional gel electrophoresis of two parent and Rif mutant strains showed identical shifts in the pI of one protein, indicating that differences between the parent and the highly Rifr mutant are not confined to the rpoB gene. These results indicate that both permeability and rpoB mutations play a role in determining the resistance of N. meningitidis to rifampin.