Equivalent MIC breakpoints to detect beta-lactamase negative ampicillin resistant Haemophilus influenzae (BLNAR) were controversial. We studied the relationship of drug resistance with gene alterations in 74 clinical isolates of H. influenzae. Out of 74 isolates, 26 showed MIC of ampicillin (ABPC) > or = 1 microg/ml. All isolates, except one, with MIC of ABPC > or = 4 microg/ml were found to produce beta-lactamase, while all 19 isolates with MIC of ABPC at 1 or 2 microg/ml were non-producing. Twenty-six ABPC resistant isolates were subjected to the analysis of genes involved in the drug resistance such as pbp3-1 pbp3-2, and TEM by the Haemophilus influenzae gene detection kit (Wakunaga Pharmaceutical Co., Ltd.) according to the supplier's instructions. Three (21.4%) of 14 beta-lactamase non-producing isolates with ABPC-MIC of 1 microg/ml had mutations of pbp3-1 gene, while all 5 non-producing isolates with ABPC-MIC of 2 microg/ml showed mutations of both pbp3-1 and pbp3-2 genes. Accordingly, it seems appropriate to set ABPC-MIC > or = 2 microg/ml for detection of BLNAR. In this study, six (8.1%) of 74 isolates were found to be BLNAR, and all of these six isolates were derived from patients of 5 year-old or younger.