Between 1996 and 2002, 103 hospitalised patients yielding one or more clinical isolates of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) were identified. A significant increase was observed in the incidence of ESBL-EC colonisation or infection during the study period (1.65 episodes/100 000 patient-days in 1996 to 12.6 episodes/100 000 patient-days in 2002; p 0.01). Infection developed in 70 (68%) patients (75 episodes), with surgical site (44%) and urinary tract (17%) infections being the most frequent. Pulsed-field gel electrophoresis showed extensive clonal diversity among the isolates. A case-control study and multivariate analysis identified female gender (OR 2.1; p 0.01), use of a nasogastric tube (OR 3.5; p 0.001) and previous antibiotic therapy (OR 3.9; p < 0.001) as independent variables associated with acquisition of ESBL-EC. The study demonstrated a progressive increase in the number of ESBL-EC isolates in a non-epidemic setting. Most cases of ESBL-EC colonisation or infection occurred in hospitalised patients exposed to invasive procedures and antibiotic pressure.