Major outbreaks of multidrug-resistant Acinetobacter baumannii associated with nosocomial infections have been increasingly reported worldwide. The endemicity of an OXA-24/40-producing A. baumannii clone associated with mortality events in Portugal has been observed at numerous hospitals within the Iberian Peninsula. Inversely, Acinetobacter haemolyticus, isolated only occasionally from clinical samples, usually presents susceptibility to different antibiotics, including β-lactams. The isolation of two carbapenem-resistant A. haemolyticus strains prompted us to assess the relative contribution of clonal spread to the observed high rate of carbapenem-resistant Acinetobacter spp. in a general hospital in Porto, Portugal. Between January 2001 and October 2004, 224 imipenem-resistant Acinetobacter spp. were collected from several specimen sources and different hospital wards. Macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and 16S rRNA gene sequencing showed that, with the exception of two clonally related A. haemolyticus isolates, the remainder were A. baumannii isolates distributed among three different pulsotypes. Clonal dissemination of two major pulsotypes (A and B), widespread throughout the hospital, contributed to the observed A. baumannii imipenem resistance, which has persisted since at least 2001 despite several elimination attempts. Clone A was identical to the previously described Iberian OXA-24/40-producing clone. Antimicrobial susceptibilities varied among isolates according to clones. Detection of carbapenemase production, ulteriorly identified as an OXA-24/40 enzyme, was positive only for clone A A. baumannii isolates and, for the first time, A. haemolyticus isolates. Hybridization assays revealed that although some clone A A. baumannii isolates showed a chromosome-positive signal for the blaOXA-24/40 probe, most also presented a positive hybridization in plasmidic bands, and similar signals were observed in A. haemolyticus isolates. We describe, for the first time, the presence of an OXA-24/40 enzyme in an A. haemolyticus clinical isolate. The observation of this enzyme in a different genomic species poses new questions on OXA-24/40 dissemination, suggesting a horizontal dissemination of the blaOXA-40 gene between different species supported by its plasmidic location. The dissemination of 'successful' clones may contribute to the high rates and persistence of imipenem-resistant A. baumannii isolates.