Antibiotics are widely useful in medicine, agriculture, and industrial fermentations. However, increasing problems with resistant strains call for restrained use and alternative strategies. Antisense peptide nucleic acids (PNAs) show potent bactericidal effects when targeted against the essential Escherichia coli acpP gene. Aside from attractive antimicrobial therapeutic possibilities for such antisense PNAs, we considered that they could be used as a substitute for antibiotics in bacterial strain selection. Here, treatment of a mixture of E. coli wild-type cells and cells carrying a binding-site altered copy of acpP (acpP-1) with anti-acpP PNA completely killed wild-type cells within 2 h, whereas cells carrying acpP-1 proliferated. Furthermore, electrotransformation of E. coli cells with the plasmid carrying acpP-1 followed by PNA selection gave rise to only true transformants. Unlike previous antibiotic-free selection strategies, this procedure does not require special growth environments or special host strains. Also, the PNA-selected cells grow at a near normal rate. The results open possibilities to use antisense PNAs for strain selection and construction in research and industrial application.