The organelles of the endocytic and autophagic pathways were studied in HeLa cells using immunoelectron microscopy and stereological techniques. In the absence of autophagic stimulation, characteristic particulate structures containing closely packed layers of membrane received the fluid-phase marker horseradish peroxidase after 25 min uptake. These multilamellar endosomes contained the majority of the cellular immunogold labeling obtained by using a monoclonal antibody (1B5) directed against a lysosomal glycoprotein. Lysosomes with homogeneous dense content were only poorly labeled. After stimulation of autophagy, two classes of autophagosome profile appeared. One had a double limiting membrane and content that resembled the surrounding cytoplasm. The other most abundant type possessed a single limiting membrane and contained multilamellar structures which were strikingly similar to multilamellar endosomes, not only in form, but also in volume and membrane packing density. Immunogold labeling showed that now the majority of 1B5 labeling was located in the class of autophagosomes which contained multilamellar structures. Stereological methods showed that, after autophagic stimulation, multilamellar endosomes had become depleted, while multilamellar structures had appeared within the autophagosomes. Taken together, these data provide evidence that autophagosomes of HeLa cells fuse with preexisting multilamellar endosomes.