Most of the organellar amino acyl-tRNA synthetases (aaRSs) are dually targeted to both mitochondria and chloroplasts using dual targeting peptides (dTPs). We have investigated targeting properties and domain structure of dTPs of seven aaRSs by studying the in vitro and in vivo import of N-terminal deleted constructs of dTPs fused to green fluorescent protein (GFP). The deletion constructs were designed based on prediction programs, TargetP and Predotar, as well as LogoPlots derived from organellar proteomes in Arabidopsis thaliana. In vitro import was performed either into a single isolated organelle or as dual import, i.e. into a mixture of isolated mitochondria and chloroplasts followed by re-isolation of the organelles. In vivo import was investigated as transient expression of the GFP constructs in Nicotiana benthamiana protoplasts. Characterization of recognition determinants showed that the N-terminal portion of TyrRS-, ValRS- and ThrRS-dTPs (27, 22 and 23 amino acids, respectively) is required for targeting into both mitochondria and chloroplasts. Surprisingly, these N-terminal portions contain no or very few arginines (or lysines), but very high number of hydroxylated residues (26-51%). For two aaRSs, a domain structure of the dTP became evident. Removal of 20 residues from the dTP of ProRS abolished chloroplastic import, indicating that the N-terminal region was required for chloroplast targeting, whereas deletion of 16 N-terminal amino acids from AspRS-dTP inhibited the mitochondrial import showing that in this case the N-terminal portion was required for the mitochondrial import. Finally, deletion of N-terminal regions of dTPs for IleRS ad LysRS did not affect dual targeting. In summary, it can be concluded that there is no general rule for how the determinants for dual targeting are distributed within dTPs but in most cases the N-terminal portion is essential for import into both organelles and in a few cases a domain structure was observed.