In selectively permeabilized fibroblasts suspended in a medium mimicking the composition of the cytosol the peroxisomal enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) was found to exhibit about 80% latency (Wolvetang, E.J., Tager, J.M. and Wanders, R.J.A. (1990) Biochem. Biophys. Res. Commun. 1035, 6-11). We investigated which components of the cytosol mimicking medium are important for latency of DHAP-AT and unmasking of latent DHAP-AT activity by ATP. We show that the latency of DHAP-AT is critically dependent upon the presence of reduced glutathione in the medium and that the in vivo prevailing GSH/GSSG ratio is sufficient to maintain DHAP-AT latency. Although thiol-groups in the peroxisomal membrane seem to be essential for the integrity of peroxisomes in selectively permeabilized fibroblasts no latency of DHAP-AT is observed in buffered sucrose media or in cell homogenates, irrespective of the presence of GSH in the medium used. We suggest that during homogenization irreversible damage is inflicted upon the peroxisomal membrane and/or that more factors than at present investigated are involved in maintaining peroxisomal integrity. Furthermore, we demonstrate that cations play a role in the stimulatory effect of ATP on latent DHAP-AT activity while a proton gradient is not directly involved in the stimulatory effect of ATP on latent DHAP-AT activity.