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We asked what peptide features govern targeting to the mitochondria versus the chloroplast using antimicrobial peptides as a starting point. This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert antimicrobial into targeting peptides, we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii. Peptides were systematically modified to test distinctive features of mitochondrial and chloroplast targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as cargo protein. Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences. Targeting to both organelles is improved by replacing Lysines with Arginines. Chloroplast targeting is enabled by the presence of flanking unstructured sequences, additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria. If indeed targeting peptides evolved from antimicrobial peptides, required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues, and a late acquisition of chloroplast specific motifs.