A model system for the study of in vitro peroxisomal protein import is described. Chinese hamster ovary (CHO) cells were permeabilized by the bacterial toxin streptolysin O (SLO) and their peroxisomes thus became accessible for a model import protein. Firefly luciferase (FL), which was previously shown to be imported into mammalian peroxisomes in vivo, was used as the reporter protein. When SLO-permeabilized CHO cells were incubated with FL in the presence of an ATP-regenerating system, import of FL could be documented by immunofluorescence staining of the cells with monospecific anti-FL antiserum. FL import increased with time and was dependent on temperature and the presence of hydrolysable ATP, which could not be replaced by GTP. The model system functioned reproducibly and should be of use for investigating fundamental questions on the mechanism of peroxisomal protein import.