The insect enzyme firefly luciferase (FL), which is known to be imported into mammalian peroxisomes as well, was introduced into the cytoplasm of chinese hamster ovary cells and human skin fibroblasts by microinjection. This model system was used to study the nucleoside triphosphate dependence of peroxisomal protein import by immunofluorescence staining of FL following depletion of cellular ATP. In energized cells a punctate staining pattern of the enzyme is observed between 30 min and 1 week after microinjection suggesting an organellar localization of FL. Evidence for its peroxisomal localization was gained by comparison of the FL staining pattern with that of catalase, a peroxisomal marker. Differential permeabilization of cells with digitonin prior to immunofluorescence staining demonstrated the intraperoxisomal localization of microinjected FL and excluded the possibility that FL is merely adhering at the cytosolic face of peroxisomes without being imported. Depletion of cellular ATP by the metabolic inhibitors 2-deoxyglucose and NaN3 completely prevented import of FL into peroxisomes whereas upon reenergizing the cells FL import was restored. The import steps that may be responsible for the observed energy dependence are discussed.