Cell-free translation products of hepatic free polysomal RNA from a clofibrate-treated rat were incubated at 26°C for 0-60 min with a post-heavy mitochondrial supernatant fraction from normal rat liver. Exogenously added proteinase K-resistant precursor and mature forms of peroxisomal 3-ketoacyl-CoA thiolase were recovered in a particulate fraction and increased with time. Both forms of thiolase cosedimented with peroxisomes, when the proteinase K-treated import reaction mixture was centrifuged in a sucrose density gradient. The in vitro import and processing of thiolase precursors, types A and B, was likewise reproduced with highly purified peroxisomes. These results strongly suggest that the precursor form of 3-ketoacyl-CoA thiolase is translocated into peroxisomes, apparently without tight coupling with proteolytic processing to the mature protein.