Peptide nucleic acid (PNA) is a DNA mimic with attractive properties for developing improved gene-targeted antisense agents. To test this potential of PNA in bacteria, PNAs were designed to target the start codon regions of the Escherichia coli beta-galactosidase and beta-lactamase genes. Dose-dependent and specific gene inhibition was observed in vitro using low nanomolar PNA concentrations and in vivo using low micromolar concentrations. Inhibition was more efficient for a permeable E. coli strain relative to wild-type K-12. The potency of the anti-beta-lactamase PNAs was abolished by a six base substitution, and inhibition could be re-established using a PNA with compensating base changes. Antisense inhibition of the beta-lactamase gene was sufficient to sensitize resistant cells to the antibiotic ampicillin. The results demonstrate gene- and sequence-specific antisense inhibition in E. coli and open possibilities for antisense antibacterial drugs and gene function analyses in bacteria.