Primers were designed to amplify the rpoB gene of Neisseria meningitidis. The region of the gene amplified covered clusters I and II of the rifampin resistance (Rifr) mutation sites identified in Escherichia coli. DNAs from six Rifr isolates and 21 rifampin-susceptible isolates from the United Kingdom representing a number of serogroups were amplified and sequenced. All six Rifr isolates had identical DNA sequences and the same amino acid change, a His to an Asn change at position 35 (H35N). This His residue is equivalent to the His residue at position 526 in E. coli, one of the known Rifr mutation sites. DNAs from an additional six Rifr mutations generated in vitro were amplified and sequenced. Three had H35Y changes, one had an H35R change, one had an H35N change and one had an S40F change. The predominance of mutations at the His residue at position 35 in Rifr N. meningitidis isolates suggests that it plays a critical role in the selection of antibiotic-resistant variants. All six Rifr isolates belonged to the same clonal group when analyzed by restriction enzyme analysis and pulsed-field gel electrophoresis. These data suggest that a single clone of Rifr N. meningitidis is present and widespread throughout the United Kingdom.