To determine the molecular basis of rifampicin resistance in Haemophilus influenzae.Mutations in the rifampicin-resistance determining region of the rpoB gene of H. influenzae were analysed by gene amplification and sequencing in 12 rifampicin resistant, one intermediate and four susceptible isolates.All clinical resistant isolates except one had at least one amino acid substitution in the beta-subunit of RNA polymerase. Eleven resistant isolates had amino acid changes at codons 513, 516, 518, 526 and 533 of cluster I, with the most common amino acid substitution being Asp-516-->Val. Only one resistant isolate also had a second mutation Asn-518-->Asp in cluster I; transformants obtained with DNA of this isolate also had both mutations. All the amino acid changes in cluster I were detected in isolates with a high level of rifampicin resistance (MIC > or =32 mg/L), except the Asp-516-->Ala mutation in a low-level resistant isolate (MIC 4 mg/L). Only one serotype f isolate with an MIC of 2 mg/L had a mutation in cluster II. Cluster III presented no amino acid changes. In in vitro-generated high-level rifampicin-resistant mutants, only amino acid changes at codons 516 and 526 were seen, with new amino acid changes appearing at codon 526 of cluster I, while His-526-->Asn was associated with low-level resistance.Rifampicin resistance in H. influenzae is due to point mutations in the rpoB gene, and the resistance levels are dependent on both the location and the nature of amino acid substitution.