Six clinical isolates of Enterobacter cloacae isolated in a Portuguese hospital, between April 1999 and November 2000, demonstrated resistance to almost all broad-spectrum cephalosporins, except to cefepime. These isolates were susceptible to quinolones and to aminoglycosides. Isoelectric focusing demonstrated production of beta-lactamases with pIs > 8.0 and by all six isolates, exhibiting a cephalosporinase phenotype. The results of pulsed field gel electrophoresis revealed that these isolates were genetically unrelated. The amino acid sequence of six AmpC beta-lactamases (Eclo1FF, Eclo6FF, Eclo9FF, Eclo10FF, Eclo11FF and Eclo15FF) shared 97-99% homology with the chromosomal AmpC beta-lactamase from E. cloacae P99 and 86-87% homology with those of two plasmid-mediated AmpC beta-lactamases, MIR-1 and ACT-1. This is the first report of chromosomal AmpC beta-lactamase production by E. cloacae isolates in a Portuguese hospital.