The import of proteins destined for the intermembrane space of chloroplasts has not been investigated in detail up to now. By investigating energy requirements and time courses, as well as performing competition experiments, we show that the two intermembrane space components Tic22 and MGD1 (E.C. 2.4.1.46) both engage the Toc machinery for crossing the outer envelope, whereas their pathways diverge thereafter. Although MGD1 appears to at least partly cross the inner envelope, Tic22 very likely reaches its mature form in the intermembrane space without involving stromal components. Thus, different pathways for intermembrane space targeting probably exist in chloroplasts.