To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).