A hybrid protein in which the immunoglobulin G-binding domain of Staphylococcus aureus protein A replaced the N-terminal 43 amino acids of glycolate oxidase (a peroxisomal protein) was affinity purified after expression in Escherichia coli and used to study peroxisomal protein import in vitro. The fusion protein, which co-purifies with the bacterial chaperones dnaK and groEL, binds to glyoxysomes and is partially translocated in an ATP-dependent reaction which is independent of eukaryotic cytosol. Both binding and translocation are dependent upon the amount of glyoxysomes present. The partially translocated species has a transmembrane location and is extractable by salt, indicating that it is held in the membrane by ionic interactions. In the absence of ATP, the fusion protein binds to the surface of the glyoxysomes and competes the binding of authentic matrix proteins. The surface-bound protein can be chased to the transmembrane species upon the addition of ATP. These results indicate that the surface-bound form is a true translocation intermediate. The availability of this fusion protein in milligram quantities offers the possibility to use the intermediate formed in the absence of ATP and the transmembrane species to probe interactions with the peroxisome import machinery.