A total of 535 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were sent to our laboratory during 1999 to 2003. All strains were analyzed by PCR to identify the beta-lactam resistance genes, and their susceptibilities to beta-lactam agents were determined. These strains were classified into 6 genotype patterns and MIC90 values for ampicillin (ABPC): (i) beta-lactamase nonproducing, ABPC susceptible (BLNAS) strains and lacked all resistance genes (27.7% of isolates; MIC90, 0.5 microg/ml); (ii) beta-lactamase producing, ABPC resistant (BLPAR) strains (12.9%, 16 microg/ml); (iii) beta-lactamase nonproducing, ABPC resistant (Low-BLNAR) strains with a Asn526Lys amino acid substitution in ftsI gene encoding PBP3 (31.2%, 2 microg/ml); (iv) beta-lactamase nonproducing, ABPC resistant (BLNAR) strains with Ser385Thr and Asn526Lys substitutions in ftsI (17.2%, 8 microg/ml); (v) amoxicillin/clavlanic acid resistant (BLPACR I) strains, having beta-lactamase gene and a Asn526Lys amino acid substitution in ftsI (9.2%, 32 microg/ml); and (vi) amoxicillin/clavlanic acid resistant (BLPACR II), having beta-lactamase gene and ftsI substitutions as for BLNAR strains (1.9%, 64 microg/ml). All but 4 strains were serotype b. The prevalence of BLNAR strains has increased exponentially: 0% (n = 0/41) in 1999, 5.8% (n = 4/69) in 2000, 14.1% (n = 19/139) in 2001, 20.1% (n = 32/159) in 2002, and 29.1% (n = 37/127) in 2003. The MIC90s of BLNAR isolates except for ABPC were as follows: piperacillin, 0.125 microg/ml; ceftriaxone, 0.25 microg/ml; meropenem, 0.5 microg/ml; cefotaxime, 1 microg/ml; panipenem, 2 microg/ml; cefozopran, 16 microg/ml; and cefotiam, 64 microg/ml. To prevent such resistance from increasing, expedited vaccination, correct identification of the BLNAR molecularly, and the proper selection of proper antibiotics based on PK/PD must be taken.