Temocillin is a narrow spectrum penicillin with high stability to most beta-lactamases including AmpC types and extended-spectrum types (ESBLs). We have analysed its in vitro activity against 652 clinical isolates of Enterobacteriaceae prospectively collected from patients hospitalised in intensive care units at seven different university hospitals in Belgium in 2005. Strains were screened for ESBL production using cefotaxime and ceftazidime screen agar plates and by double ESBL E-tests. The MIC of temocillin and of five comparators was determined using the E-test method. ESBLs were characterized at one central laboratory by isoelectric focusing, PCR for bla genes of the SHV, TEM, and CTX-M families, and by DNA sequencing. The prevalence of ESBL-producing Enterobacteriaceae averaged 11.8% and ranged between 3.0 and 29% in the different hospitals. Meropenem exhibited the highest in vitro activity overall (mode MIC 0.064 microg; MIC(90); 0.19 microg/ml), whereas ceftazidime (MIC(90) > 256 microg/ml) and ciprofloxacin (MIC(90) > 32 microg/ml) scored the worst. Temocillin was active against more than 90% of the isolates including most AmpC- and ESBL-producing isolates. These data indicate the well preserved activity of temocillin over the years against Enterobacteriaceae and show the wide variation in prevalence of ESBL-producing Enterobacteriaceae isolates in Belgian intensive care units. Prospective clinical studies are, however, needed to validate the usefulness of temocillin in the treatment of microbiologically documented infections caused by ESBL- and/or AmpC- overproducing nosocomial Enterobacteriaceae pathogens.