cPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF) 3 RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is ␤-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF) 3 RXB-AcpP was 2.5 M (14 g/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF) 3 RXB-AcpP was 4 ؋ 10 ؊7 mutations/cell division. A spontaneous (RFF) 3 RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF) 3 RXB-AcpP was 40 M (224 g/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF) 3 RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF) 3 RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF) 3 RXB-AcpP. Deletion of sbmA caused resistance to (RFF) 3 RXB-AcpP. We conclude that resistance to (RFF) 3 RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF) 3 R-XB PPMOs may be transported across the plasma membrane by SbmA.