Objective To study the method for phenotype detection of AmpC beta-lactamase from Klebsiella pneumoniae and Escherichia coli,and to investigate the distribution of AmpC beta-lactamase and extended spectrum beta-lactamase (ESBL) in Klebsiella pneumoniae and Escherichia coli which were unsusceptible to cefoxitin.Methods AmpC beta-lactamase from Klebsiella pneumoniae and Escherichia coli were detected by 3-aminophenylboronic acid (APB) disk potentiation test in comparison with Tris-EDTA disk method. Ceftazidim,cefotaxim and cefoxitin with and without APB were used in the APB disk potentiation test.The disk confirmatory test for detection of ESBL approved by NCCLS was used for detection of ESBL.Results The APB disk potentiation test got good agreement with Tris-EDTA disk method. By detection using both methods 41 were AmpC beta-lactamase-producing in 62 strains of Klebsiella pneumoniae unsusceptible to cefoxtin (accounted for 66.1%) and 33 were AmpC beta-lactamase-producing in 74 strains of Escherichia coli unsusceptible to cefoxtin (accounted for 45.0%) .In Klebsiella pneumoniae,AmpC beta-lactamase with ESBL together was very common. AmpC beta-lactamase only,ESBL only and AmpC beta-lactamase with ESBL accounted for 4.5%,21.0% and 51.6% respectively.However, in Escherichia coli,ESBL only,ESBL with AmpC beta-lactamase,and AmpC beta-lactamase only accounted for 40.5%,24.3% and 20.3% respectively.Conclusion Both APB disk potentiation test and Tris-EDTA disk test were easy,convenient and cheap methods which could be applied for detection of AmpC beta-lactamase from Klebsiella pneumoniae and Escherichia coli in clinical laboratory.