The affinity of [3H]-benzylpenicillin for penicillin-binding protein(PBP) 3A/3B was reduced in clinical isolates beta-lactamase-negative ampicillin(ABPC)-resistant Haemophilus influenzae(BLNAR) with MIC of > or = 1 microgram/ml to ABPC. The sequence of ftsI gene encoding the transpeptidase domain of PBP3A/3B were determined for these strains, and compared to those of ABPC-susceptible Rd strain. Common substitutions of deduced amino acid residues were identified in transpeptidase region on the ftsI gene in BLNAR strains. Homology modeling of the three-dimensional structure of PBP3 showed that every common substitution occurred at active site pocket surrounded by three conserved motifs. The MICs of beta-lactams for H. influenzae transformants in which ftsI gene from BLNAR was introduced, were as high as those for the donors and PBP3A/3B showed a decreased affinity for beta-lactams. These data indicate that mutations in the ftsI gene are the most important for development of resistance to beta-lactams in BLNAR.