The penicillin binding protein (PBP) genes dacA, dacB and ftsI from 14 cefuroxime-resistant (CXM(R)) isolates and three clinical isolates with low CXM MIC for non-beta-lactamase-producing Haemophilus influenzae type b were molecularly characterized. One strain, 5788, was used to transform H. influenzae Rd to CXM(R) for direct comparison of the pbps in the same genetic background. No obvious mutations in the dacA and dacB gene products could be associated with CXM(R). One amino acid substitution in the ftsI gene product in particular, S357N, could give rise to CXM(R). Sequence analysis from the CXM(R) transformants also implicated FtsI; in this case, the substitutions were V511A and R517H. To verify S357N substitution, the protein sequence of H. influenzae FtsI was threaded through the S. pneumoniae PBP 2X structure giving an average root mean square deviation of the alpha-carbon chains of 0.5 A. The S357N substitution alters both the residue size and charge. One explanation for the contribution of S357N to CXM(R) is that the asparagine side-chain produces unfavourable steric hindrance with the side chain of Val-362 changing the torsion angles of the asparagine residue, which in turn may influence the position of the loop V362-P366 adjacent to the active site. Whilst other groups have examined the contribution of H. influenzae PBPs in ampicillin resistance, this is the first report analysing their role in CXM(R).