The targeting mechanism of chloroplast outer envelope membrane proteins remains largely unknown. We investigated the targeting of AtToc64. In protoplasts, the transmembrane domain (TMD) and its C-terminal Iysine-rich flanking region (LFR) were both necessary and sufficient for targeting to the outer envelope membrane. The lysine residues of the flanking region were critical; without the LFR, the TMD was targeted to the ER or the plasma membrane. In addition, the types of amino acid residues of the TMD, but not the amino acid sequence per se, is a signal for targeting to the chloroplast envelope membrane. TMDs containing phenylalanines were not targeted to the chloroplast in vivo. Based on these results, we propose that the chloroplast targeting signal of AtToc64 comprises two different components: 1) the LFR, which is a signal for evading SRP-mediated co-translational translocation and 2) the hydrophobic amino acid side chains of the TMD, whose size functions as a signal for a cytosolic factor that mediates transport to the chloroplast.