We analyzed the incidence of beta-lactamase non-producing ampicillin-resistance (BLNAR) Haemophilus influenzae in Showa University Hospital from June 2004 to March 2005. The ratio of BLNAR in total H. influenzae isolates was 44.5%. BLNAR accounted for 89.6% of the ampicillin non-susceptible strains displaying a minimal inhibitory concentration(MIC) of ampicillin of more than 2 microg/ml, which is the break point advocated by the Clinical and Laboratory Standards Institute. Of the total BLNARs, 71.4% were isolated from children of six and under. We analyzed mutations of the SSN and KTG motif of penicillin binding protein 3 encoded by the ftsI gene, since mutations in these regions are considered to be responsible for the drug-resistance of BLNAR. Mutations in SSN and KTG motifs were identified in all BLNAR isolates. We also detected mutations in the ampicillin susceptible strains displaying an ampicillin MIC of 0.5 or microg/ml. When a noticeable MIC increase of ABPC or other beta-lactams in the routine clinical laboratory practice, gene analysis of ftsI gene of the isolates displaying increased MIC is required to measure the spreading of BLNAR.