The transport of glycerolipid intermediates, viz. palmitoyl dihydroxyacetone phosphate (DHAP) and lysophosphatidate from peroxisomes and their conversion to phosphatidate in endoplasmic reticulum (microsomes) were studied in cell-free systems. The lipids were biosynthesized from [32P]DHAP, palmitoyl-CoA, and freshly made rat liver peroxisomes and microsomes in the presence or absence of Mg2+, NADPH, and bovine serum albumin (BSA). After incubation, the soluble fraction and the membranes were separated, and the distribution of radioactive lipids in these fractions were determined. The results showed that palmitoyl-DHAP and lysophosphatidate were recovered in the supernatant when BSA was present or when BSA was absent, but Mg2+ was removed after incubation by chelation with EDTA (or ATP). At low optimum palmitoyl-CoA concentration or when palmitoyl-CoA was generated in peroxisomes, and in the absence of BSA, the biosynthesized keto ether and ester lipids and lysophosphatidate were similarly present in the supernatant. Phosphatidate, however, was always localized in the membranes. Further fractionation showed that phosphatidate was associated with the microsomes. The critical micellar concentrations of palmitoyl-DHAP and 1-palmitoyl-rac-glycerol 3-phosphate, under the incubation conditions used, were determined to be 58 and 70 microM, respectively. These results suggest that at physiological concentrations the biosynthesized lysolipids are water soluble, and therefore, a carrier protein is unnecessary for their transport. These lipids freely diffuse from peroxisomes to endoplasmic reticulum where they are converted to membrane-bound phosphatidate.