The enzyme, dihydroxyacetone-phosphate acyltransferase (DHAP-AT) is localized at the inner surface of the peroxisomal membrane but shows no latency. The product, i.e., acyl-DHAP, is the first lipidic intermediate in ether lipid biosynthesis that is converted by alkyldihydroxyacetone-phosphate synthase into alkyl-DHAP. This step and the reductions of acyl-DHAP and alkyl-DHAP to the glycerol 3-phosphate analogs can still take place in peroxisomes. The further conversions of these intermediates into ether-linked choline or ethanolamine glycerophospholipids or plasmalogens take place in the endoplasmic reticulum. In this paper we describe studies to localize acyl-DHAP in the transversal plane of the peroxisomal membrane. To enable these studies, the usually employed assay conditions for DHAP-AT were modified by omission of BSA and by lowering the temperature and the palmitoyl-CoA concentration. Using these modified conditions, we were able to label peroxisomes with endogenously generated acyl-DHAP, with retention of catalase within the peroxisomal membrane. Endogenously generated acyl-DHAP was rapidly extractable from the outer surface of peroxisomes with BSA at 0 degree C, suggesting that even at this temperature the product was transported very fast across the membrane. Trypsin treatment of peroxisomes did not affect this behaviour. Only after short incubation periods, an increase in the proportion of non-extractable acyl-DHAP was observed. In large unilamellar vesicles made from peroxisomal phospholipids no transmembrane movement of acyl-DHAP was found. Despite the apparently rapid transbilayer movement of acyl-DHAP in the presence of BSA, it could still serve as a substrate for the enzyme alkyl-DHAP synthase, which is also localized at the inner surface of the peroxisomal membrane. In addition it was shown that endogenously generated substrate is used at a higher efficiency compared to exogenously added acyl-DHAP, suggesting a close interaction of the two enzymes in the peroxisomal membrane.