Biosynthesis and intracellular transport of 70-kda peroxisomal membrane protein (pmp70) has been studied in rat hepatoma, h-4-ii-e cells. Pulse-chase analysis showed that a newly synthesized 35S-PMP70 first appeared in the cytosolic fraction and was then transported into the peroxisomal fraction. The half-life of 35S-PMP70 in the cytosolic fraction was approximately 3 min. Integration of 35S-PMP70 into membranes occurred in the peroxisomal fraction and was completed within 30 min. No proteolytic processing of 35S-PMP70 was observed. An in vitro import system was reconstituted to characterize the insertion mechanism of PMP70 into peroxisomes. Peroxisomes isolated from rat liver were incubated at 26 degrees C with [35S]methionine-labeled in vitro translation products of PMP70 mRNA in the presence of the cytosolic fraction. The peroxisomes were reisolated and insertion of 35S-PMP70 into the membrane was analyzed using a Na2CO3 procedure. The binding and insertion of 35S-PMP70 were dependent on temperature and incubation time and was specific for peroxisomes. Pretreatment of peroxisomes with trypsin and chymotrypsin almost abolished the binding and insertion of 35S-PMP70. The translation products contained several truncated 35S-PMP70s. The NH2 terminally truncated 35S-PMP70s, with a molecular mass greater than 50 kDa, bound to and inserted into peroxisomal membranes, whereas truncated 35S-PMP70s smaller than 45 kDa did not. These results suggest that PMP70 is post-translationally transported to peroxisomes without processing and inserted into peroxisomal membranes by a specific mechanism in which a proteinaceous receptor and a certain internal sequence of PMP70 are involved.