Apidaecins, short proline-arginine-rich peptides from insects, are highly bactericidal through a mechanism that includes stereoselective elements but is completely devoid of any pore-forming activity. The spectrum of antibacterial activity, always limited to Gram-negatives, is further dependent on a small number of variable residues and can be manipulated. We show here that mutations in the evolutionary conserved regions result in a more general loss of function, and we have used such analogs to probe molecular interactions in Escherichia coli. First, an assay was developed to measure selectively chiral association with cellular targets. By using this method, we find that apidaecin uptake is energy-driven and irreversible and yet can be partially competed by proline in a stereospecific fashion, results upholding a model of a permease/transporter-mediated mechanism. This putative transporter is not the end point of apidaecin action, for failure of certain peptide analogs to kill cells after entering indicates the existence of another downstream target. Tetracycline-induced loss of bactericidal activity and dose-dependent in vivo inhibition of translation by apidaecin point at components of the protein synthesis machinery as likely candidates. These findings provide new insights into the antibacterial mechanism of a unique group of peptides and perhaps, by extension, for distant mammalian relatives such as PR-39.