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This study aimed to evaluate the metallo-beta-lactamase (MBL) production in Pseudomonas spp. and Acinetobacter baumannii using phenotypic and genotypic methods and to determine the most appropriate phenotypic method. The study included 55 Pseudomonas spp. (53 Pseudomonas aeruginosa, 1 P. fluorescens and 1 P. putida) and 33 A. baumannii isolates which were resistant to imipenem (IMP) and/or meropenem (MEM). Six phenotypic and one genotypic (real-time polymerase chain reaction [RT-PCR]) methods were used. According to the phenotypic tests, the rates of MBL-positive Pseudomonas spp. and A. baumannii were, respectively: 25.5% and 39.4% by the gradient test; 21.8% and 21.2% by the Rosco rapid CARB screen test; 9.1% and 21.2% by the modified Hodge test (MHT); 32.7% and 66.7% by the combined EDTA disk diffusion test; 56.4% and 100% by IMP + EDTA and 49.0% and 72.7% by MEM + EDTA and 9.1% and 3.0% by IMP + dipicolinic acid (DPA) for the Rosco MBL confirm test; 36.4% and 6.1% by IMP + DPA and 54.5% and 6.1% MEM + DPA for the double disk synergy test. MBL genes were detected only in three Pseudomonas spp. (blaIMP in two P. aeruginosa isolates and blaVIM in a P. fluorescens isolate). For Pseudomonas spp., the MBL positivity rate did not significantly differ between the RT-PCR and MHT and between the RT-PCR and Rosco MBL confirm test (with IMP + DPA) (p > 0.10). In conclusion, the Rosco MBL confirm test (with IMP + DPA) phenotypically predicted the MBL positivity most closely to the RT-PCR method for both Pseudomonas spp. and A. baumannii isolates. © 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.