An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S-Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions approximately 30% of [35S]AOx became proteinase resistant. The import of AOx into peroxisomes could be dissociated into two steps: (a) binding occurred at 0 degrees C in the absence of ATP; (b) translocation occurred only at 26 degrees C and required the hydrolysis of ATP. GTP would not substitute for ATP and translocation was not inhibited by carbonylcyanide-m-chlorophenylhydrazone, valinomycin, or other ionophores.

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