OBJECTIVE: To investigate the situation of antibiotic resistance of Haemophilus influenzae isolates from Chinese children. METHODS: Eight hundred and ninety-eight strains of H. influenzae isolated from randomly selected pediatric out-patients in Beijing, Shanghai and Guangzhou 2000 approximately 2002 underwent determination of antibiotic susceptibility by E test MIC method for beta-lactam antibiotics (ampicillin, amoxicillin/clavulanic acid, ceftriaxone, cefuroxime, and cefaclor) and KB disc diffusion method for chloramphenicol, tetracycline, sulfamethoxazole/trimethoprim (SMZ/TMP), azithromycin, and ciprofloxacin. RESULTS: The average rate of beta-lactamase production among these isolates was 12%. The MIC50 and MIC90 of ampicillin were 0.25 microg/ml and 4 microg/ml respectively for the 898 isolates, 88% of the isolates were susceptible to ampicillin and 10.6% was resistant. There was no beta-lactamase negative strains among the ampicillin intermediately-susceptible and resistant strains. All the isolates were susceptible to amoxicillin/clavulanic acid, ceftriaxone, cefuroxime and azithromycin. 96.1% of the H. influenzae isolates generally remained susceptible to cefaclor with a MIC50 of 1.5 microg/ml and a MIC90 of 3 microg/ml, and 99% of the isolates were susceptible to ciprofloxacin. The susceptibility rate to tetracycline of the isolates from Beijing and Shanghai areas were 57% and 61% respectively, both lower than the susceptibility rates of the isolates from Guangzhou (81%), while the resistant rate to SMZ/TMP of the isolates from Beijing was 35%, lower than those of the isolates from Guangzhou (47%) and Shanghai (54%). The resistant rate to ampicillin was 7.7% in 2000 and was increased to 14.5% in 2002. 34.5% of all the isolates were susceptible to 8 antimicrobial agents while 12.8% was multi-drug resistant. 58.5% of the isolates were distributed in five major antimicrobial resistance profiles in which SMZ/TMP resistance (29.8%) was the most prevalent, followed by SMZ/TMP and tetracycline combined resistance (12.5%) and tetracycline resistance (9.4). The ampicillin non-susceptible isolates had higher resistant rates to cefaclor (23.5%), tetracycline (63.3%), SMZ/TMP (74.5%) and chloramphenicol (63.3%) than the ampicillin susceptible strains (1.6%, 51.4%, 11.1% and 4.9% respectively). CONCLUSION: Beta-lactamase production and ampicillin resistance among the isolates from Chinese children are increasing generally during the period 2000 approximately 2002. There is variation of resistance among different regions. There are strong correlations between ampicillin resistance and resistance to cefaclor, chloramphenicol, and tetracycline among the H. Influenzae isolates.