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Objective:To determine the relationship between the application of antibiotic and the production of extended spectrum β-lactamases (ESBLs).Methods:ESBLs were detected by the double disc diffusion method among 161 gram-negative bacilli isolated from clinical specimens.The use of antibiotic in the patients was investigated before ESBLs were detected. Results:The rate of ESBLs-producing strains was 35.4% (57/161). In the patients with ESBLs-producing strains, the utilization rates of antibiotic and the third-generation cephalosprin were 96.4% (55/57) and 42.1% (24/57) respectively, which were significantly higher than those without ESBLs-producing strains [80.6% (84/104) and 16.3% (17/104), respectively].There were significant differences (P<0.05 and P<0.005,respectively) between the two groups of patients.The duration of the third-generation cephalosprins therapy was significantly longer in the patients with ESBLs-producing strains [(7.5 ± 5.4)d] than those without ESBLs-producing strains [(2.7±2.6)d,P<0.05].Conclusion:The application of antibiotic, especially the third-generation cephalosprins, is the risk factor for the production of ESBLs.