Peroxisomal matrix proteins are posttranslationally imported into peroxisomes with the peroxisome-targeting signal 1 receptor, Pex5. The longer isoform of Pex5, Pex5L, also transports Pex7-PTS2 protein complexes. After unloading the cargoes, Pex5 returns to the cytosol. To address molecular mechanisms underlying Pex5 functions, we constructed a cell-free Pex5 translocation system with a postnuclear supernatant fraction from CHO cell lines. In assays using the wild-type CHO-K1 cell fraction, (35)S-labeled Pex5 was specifically imported into and exported from peroxisomes with multiple rounds. (35)S-Pex5 import was also evident using peroxisomes isolated from rat liver. ATP was not required for (35)S-Pex5 import but was indispensable for export. (35)S-Pex5 was imported neither to peroxisome remnants from RING peroxin-deficient cell mutants nor to those from pex14 cells lacking a Pex5-docking site. In contrast, (35)S-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, (35)S-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export. Moreover, we analyzed the (35)S-Pex5-associated complexes on peroxisomal membranes by blue-native polyacrylamide gel electrophoresis. (35)S-Pex5 was in two distinct, 500- and 800-kDa complexes comprising different sets of peroxins, such as Pex14 and Pex2, implying that Pex5 transited between the subcomplexes. Together, results indicated that Pex5 most likely enters peroxisomes, changes its interacting partners, and then exits using ATP energy.