Plastid envelope DNA-binding protein (PEND) is a DNA-binding protein with a chloroplast basic region-zipper domain at its N-terminus and a transmembrane domain at its C-terminus. The localization of PEND to the inner envelope membrane was demonstrated in a targeting experiment using isolated membranes and green fluorescent protein-tagged fusion proteins. An N-terminal sequence analysis showed that the presequence is 15 amino acids long; however, based on neural network-based prediction tools, this short peptide is not predicted to be a chloroplast-targeting sequence. In the present study we confirmed, by the digestion of intact chloroplasts, that PEND is located in the envelope membrane. We then demonstrated that the N-terminal 88-amino acid sequence is sufficient for plastid import in vitro. The transient expression of green fluorescent protein-tagged fusion proteins revealed that neither the N-terminal 29-amino acid sequence nor the 16-amino acid sequence directed green fluorescent protein to chloroplasts, but that the N-terminal 66-amino acid sequence was sufficient for correct targeting. These results suggest that targeting of PEND to the chloroplast requires both the presequence and the basic region, whereas postimport processing cleaves only the presequence. Interestingly, deletion of the presequence in the green fluorescent protein-tagged 88-amino acid construct resulted in targeting to the nucleus. This raises the possibility of plastid-to-nuclear signal transduction by the relocalization of PEND.