Toc75 is the channel for protein translocation across the chloroplast outer envelope membrane. Toc75 belongs to the Omp85 protein family and consists of three N-terminal polypeptide transport-associated (POTRA) domains that are essential for the functions of Toc75, followed by a membrane-spanning β-barrel domain. In bacteria, POTRA domains of Omp85 family members are located in the periplasm, where they interact with other partner proteins to accomplish protein secretion and outer membrane protein assembly. However, the orientation and therefore the molecular function of chloroplast Toc75 POTRA domains remain a matter of debate. We investigated the topology of Toc75 using bimolecular fluorescence complementation and immunogold electron microscopy. Bimolecular fluorescence complementation analyses showed that in stably transformed plants, Toc75 N terminus is located on the intermembrane space side, not the cytosolic side, of the outer membrane. Immunogold labeling of endogenous Toc75 POTRA domains in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana) confirmed that POTRA domains are located in the intermembrane space of the chloroplast envelope.