To determine whether the widespread clinical use of -lactams has been selective for Citrobacter freundiiderived alleles of plasmid ampC genes, we generated a Bayesian consensus phylogeny of the published ampC sequences and compared the MICs of 16 -lactam antibiotics for Escherichia coli strains containing cloned copies of the C.freundii ampC alleles. We found that for the majority of compounds investigated, there has been essentially no increase in beta-lactam resistance conferred by those alleles. We also found that ampC alleles from the chromosomes of two beta-lactam-sensitive C.freundii strains isolated in the 1920s, before the clinical use of antibiotics, were as effective at providing beta-lactam resistance in E. coli as were the plasmid-borne alleles from beta-lactam-resistant clinical isolates. These results suggest that selection for increased resistance to beta-lactam antibiotics has not been a significant force directing the evolution of the C.freundii ampC alleles found in beta-lactam-resistant clinical isolates.