Objective To study the resistance mechanism of ampicillin-resistant Haemophilus influenzae strains collected from hospitals in Beijing,Shanghai and Guangzhou during the past four years.Methods During 2000—2003,899 strains of H.influenzae isolated from children with respiratory tract infection were collected to detect their resistance to ampicillin.The susceptibility testing was determined by E test.β-lactamase production was detected by chromogenic cephalosporin (nitrocefin) test.TEM-1 and ROB-1 gene conferring ampicillin resistance were detected by polymerase chain reaction (PCR).Results β-lactamase was produced in all the 74 ampicillin-resistant H.influenzae strains.They all contained TEM-1 gene.No ROB-1 gene was detected in these isolates.Conclusions TEM-1 β-lactamase is the principal mechanism of ampicillin resistance of H.influenzae isolates from the upper respiratory tract of children.