Certain enzymes normally associated with peroxisomes, such as the dihydroxyacetone phosphate (DHAP) acyltransferase involved in plasmalogen biosynthesis, are present at low levels in peroxisome-deficient mutants of Chinese hamster ovary (CHO) cells. We now show that the aminoglycoside G418 increases the residual DHAP acyltransferase in mutant ZR-82 by 60-fold. This is accompanied by a dose- and time-dependent restoration of the plasmalogen content. G418 treatment of ZR-82 also increases residual peroxisomal beta-oxidation activity by 3.8-fold. G418 does not affect wild-type CHO cells (CHO-K1) or a different peroxisome-deficient mutant, ZR-78.1. The effects of G418 on ZR-82 are transient, since plasmalogens and DHAP-acyltransferase decline to basal levels 5 days after G418 withdrawal. Other aminoglycosides and lysosomotropic agents do not alter plasmalogen levels in ZR-82. The subcellular distribution of catalase (an enzyme of the peroxisomal matrix which is present in normal amounts in peroxisome-deficient mutants but is mislocalized in the cytosol) is unaffected by G418 treatment of ZR-82, demonstrating that G418 does not restore peroxisomes. Localization of catalase by immunofluorescence microscopy confirms a total absence of intact peroxisomes in ZR-82, either before or after exposure to G418. This study is the first to demonstrate that some peroxisome-deficient mutants can be induced to accumulate functional DHAP acyltransferase and other peroxisomal enzymes, usually missing in the absence of peroxisomes. G418 may have some therapeutic value in selected patients with inborn errors of peroxisome assembly, such as Zellweger syndrome.