This study describes the first two reported invasive nontypeable Haemophilus influenzae (NTHI) isolates (strains 183 and 184) with heterogeneous resistance to imipenem. For both isolates, Etest showed imipenem MICs of > or =32 microg/ml. When the two strains were examined by the quantitative method of population analysis, both strain populations were heterogeneously resistant to imipenem and contained subpopulations growing in the presence of up to 32 microg of imipenem/ml at frequencies of 1.7 x 10(-5) and 1.5 x 10(-7), respectively. By pulsed-field gel electrophoresis analysis, the two isolates appeared to be genetically closely related. The sequencing of the ftsI gene encoding penicillin-binding protein 3 (PBP 3) and comparison with the sequence of the imipenem-susceptible H. influenzae strain Rd identified a pattern of six amino acid substitutions shared between strains 183 and 184; an additional change was unique to strain 183. No relationship between mutations in the dacB gene encoding PBP 4 and imipenem resistance was found. The replacement of the ftsI gene in the imipenem-susceptible strain Rd (for which the MIC of imipenem is 0.38 to 1 microg/ml) with ftsI from strain 183 resulted in a transformant for which the MIC of imipenem ranged from 4 to 8 microg/ml as determined by Etest. The Rd/183 transformant population showed heterogeneous resistance to imipenem; it contained subpopulations growing in the presence of up to 32 mug of imipenem/ml at a frequency of 3.3 x10(-8). The presence of additional resistance mechanisms, such as the overexpression of the AcrAB efflux pump, was investigated and does not seem to be involved. These data indicate that the heterogeneous imipenem resistance phenotype of our NTHI clone depends largely on the PBP 3 amino acid substitutions. We speculated that bacterial regulatory networks may play a role in the control of the heterogeneous expression of the resistance phenotype.