A predictive approach was assayed to evaluate the possibility of mutant Amp-C beta-lactamase emergence with increased substrate spectrum (including new C-3' quaternary ammonium cephems). The ampC gene encoding the AmpC beta-lactamase from Enterobacter cloacae was cloned and expressed in an AmpC-defective strain of E. coli. After the AmpC containing strain was challenged with cefpirome, an ampC variant encoding an enzyme with increased resistance to cefpirome and cefepime was selected. In addition, this variant conferred increased resistance to penicillins and third generation cephalosporins. The complete nucleotide sequence of the gene was determined. The deduced peptide sequence showed a single change with respect to the wild-type gene: valine to glutamic acid at position 318 of the native protein (298 of the mature enzyme). The potential emergence and spread of this type of AmpC variants among pathogens should be considered.