The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/microm2, whereas isolated lysosomes had about 2000 particles/microm2). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/microm2), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearence of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi- or multilamellar autophagic vacuoles appeared to engage in such fusions.