The sequestration and delivery of cytoplasmic material to the yeast vacuole and mammalian lysosome require the dynamic mobilization of cellular membranes and specialized protein machinery. Under nutrient deprivation conditions, double-membrane vesicles form around bulk cytoplasmic cargo destined for degradation and recycling in the vacuole/lysosome. A similar process functions to remove excess organelles under vegetative conditions in which they are no longer needed. Biochemical, morphological, and molecular genetic studies in yeasts and mammalian cells have begun to elucidate the molecular details of this autophagy process. In addition, the overlap of macroautophagy with the process of pexophagy and with the biosynthetic cytoplasm-to-vacuole targeting pathway, which delivers the resident vacuolar hydrolase aminopeptidase I, indicates that these three pathways are related mechanistically. Identification and characterization of the autophagic/cytoplasm-to-vacuole protein-targeting components have revealed the essential roles for various functional classes of proteins, including a novel protein conjugation system and the machinery for vesicle formation and fusion.