Carbapenem-hydrolysing oxacillinases are reported increasingly in Acinetobacter baumannii. This study investigated the role of these β-lactamases in causing resistance to carbapenems in 83 epidemiologically related and unrelated imipenem-resistant A. baumannii clinical isolates. The isolates were also analysed for the presence of ISAba1 in the promoter region of the blaOXA-51-like gene in order to investigate the role of ISAba1 in OXA-51 expression. All clinical isolates contained a blaOXA-51-like gene, 20% contained a blaOXA-58-like gene, and 42% contained a blaOXA-40-like gene; blaOXA-23-like, blaIMP and blaVIM genes were not detected in any of the isolates investigated. ISAba1 was found in 24 (82.7%) of 28 pulsetypes, and was located in the promoter region of the blaOXA-51-like gene in five (20.8%) of these pulsetypes. Expression of blaOXA-51 was detected in the five isolates with ISAba1 located in the promoter region, but was not detected in an isogenic imipenem-susceptible A. baumannii isolate that did not have ISAba1 located in the promoter region. It was concluded that there is a high prevalence of oxacillinases with activity against carbapenems among genetically unrelated A. baumannii clinical isolates from Spain, and that concomitant expression of two carbapenemases (OXA-51-like and either OXA-40-like or OXA-58-like) may take place. Insertion of an ISAba1-like element in the promoter of the blaOXA-51-like gene promotes the expression of this gene, although this did not seem to play a major role in carbapenem resistance.