Objective This study was designed to investigate ampicillin resistance in H.influenzae strains isolated from infected children in Suzhou,and the mechanism of resistance.Methods The susceptibility of 135 of clinical isolates of H.influenzae were tested by Kirby-Bauer disc method.Chromogenic nitrocephin disc was used to detect beta-lactamase.TEM and ROB beta-lactamase genes were detected by PCR.Results The rate of ampicillin resistance was 17.8% in H.influenzae isolated from children in Suzhou.Beta-lactamase was produced by all ampicillin-resistant H.influenzae isolates.No beta-lactamase negative ampicillin-resistant H. influenzae (BLNAR) was identified.Beta-lactamase gene TEM was detected in 27.4% of H.influenzae isolates (TEM gene was also present in 10.4% of ampicillin-sensitive strains).ROB gene was detected in 0.7% of H.influenzae isolates (This ROB-type strain also carried TEM gene).One strain of non-TEM-type,and non-ROB-type beta-lactamse-producing H.influenzae was identified.Conclusions Ampicillin resistance in H.influenzae isolates from children in this region is challenging.The resistance mechanism of ampicillin resistant H.influenzae is production of beta-lactamase,mainly TEM-type enzyme.It should be noted that the emergence of ampicillin-sensitive strain carrying TEM gene,which may predicts the development of new ampicillin-resistant strains.