Pseudomonas cepacia 249 produces an inducible beta-lactamase with penicillinase activity. The nucleotide sequence of the penA gene, which encodes this beta-lactamase, was determined and found to include regions with a significant homology to the ampC-encoded beta-lactamases of members of the family Enterobacteriaceae and Pseudomonas aeruginosa. The predicted amino acid sequence of the PenA beta-lactamase contained 17 amino acids immediately preceding the putative active-site serine which were highly conserved among the enzymes of the AmpC family. Although the penA-coding sequence had a total GC content of 60%, the predicted codon usage was more characteristic of Escherichia coli ampC-encoded beta-lactamase, with 53% of the codons having G or C in the third position, in contrast to the values for the P. aeruginosa ampC (88.5%) or Pseudomonas cepacia (88 to 92%) metabolic genes. The inducible expression of penA can be regulated by the E. coli gene product AmpD. A putative P. cepacia AmpR homolog was associated with the positive regulation of both Enterobacter cloacae ampC and P. cepacia penA expression, as confirmed by gel retardation studies. The E. cloacae AmpR did not regulate penA expression. Thus, by homology studies, codon usage, and genetic analysis, the P. cepacia penA beta-lactamase appears to have been acquired from members of the family Enterobacteriaceae and belongs to the class C group of beta-lactamases.