Microsomal membranes from rat liver were treated with the cross-linking reagent 1,5-difluoro-2,4-dinitrobenzene (DFDNB). Experimental work showed that at a probe concentration of 0.75 mM all free phosphatidylethanolamine (PE) and phosphatidylserine (PS) were found as dinitrophenyl derivatives: 29% of PE was in monomeric form, 9% dimeric, 2% interacted with PS, and 63% cross-linked to protein. PS showed a greater percent in monomeric and dimeric form and only 31% was cross-linked to protein. The cross-linking pattern of PE was clearly different from that pattern which is present in the inner mitochondrial and erythrocyte membranes. In vivo labeling of PE with [(3)H]glycerol and [(3)H]ethanolamine followed by phospholipase A(2) treatment of isolated microsomes established a heterogeneous labeling pattern during the first 2 hours. During this period, the specific activity of the phospholipase A(2)-sensitive compartment was considerably higher. The differential distribution of radioactivity after in vivo labeling in the part of the PE which reacted with increasing concentrations of DFDNB also indicated compartmentalization. After in vivo labeling with the precursors, the time course of the specific radioactivity demonstrated an initial high labeling, almost exclusively in the monomeric form, followed by a later appearance of the label in the protein-bound PE. The experiments indicate that the biosynthesis of PE takes place in a compartment that is more accessible to surface probes and that the labeled molecules are transferred in a time-dependent process to a second compartment where the lipid is not available for phospholipase A(2) action but is available for cross-linking to protein.-Valtersson, C., and G. Dallner. Compartmentalization of phosphatidylethanolamine in microsomal membranes from rat liver.