Objectives: To determine the distribution of acquired AmpC β-lactamases in 173 isolates of Escherichia coli and Klebsiella spp. submitted to the UK's national reference laboratory for antibiotic resistance. Methods: MICs were determined and interpreted according to BSAC guidelines. Candidate isolates were those resistant to cefotaxime and/or ceftazidime, irrespective of addition of clavulanic acid. Genes encoding six phylogenetic groups of acquired AmpC enzymes were sought by PCR. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE), and one blaAmpC amplicon was sequenced. Results: Genes encoding acquired AmpC enzymes were detected in 67 (49%) candidate E. coli and 21 (55%) Klebsiella spp. Sixty isolates produced CIT-type enzymes, 14 had ACC types, 11 had FOX types and 3 had DHA enzymes. The low-level cephalosporin resistance of the remaining isolates (n = 85; 49%) was inferred to result from reduced permeability or, in E. coli, from hyperexpression of chromosomal ampC. Twenty-four E. coli isolates from one hospital produced a CIT-type enzyme, with 20 of these additionally producing a group 1 CTX-M extended-spectrum β-lactamase. PFGE indicated that these isolates belonged to UK epidemic strain A, which normally produces CTX-M-15, but no acquired AmpC. Sequencing a representative bla AmpC amplicon indicated that in one centre this strain had acquired a novel CMY-2 variant, designated CMY-23. Conclusions: Diverse acquired AmpC enzymes occur in E. coli andKlebsiella spp. isolates in the UK and Ireland, with CIT types the most common. Producers are geographically scattered, but with some local outbreaks. Acquisition of a CMY-2-like enzyme by E. coli epidemic strain A suggests that these enzymes may be poised to become an important public health issue.