Many organelles change their shape in the course of the cell cycle or in response to environmental conditions. Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary membrane and the subsequent scission of vesicles into the lumen of the organelle. The mechanism driving these structural changes is enigmatic. We have begun to analyze this process by reconstituting microautophagocytosis in a cell-free system. Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion. During the uptake reaction, vacuolar membrane invaginations, called autophagic tubes, were observed. The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation. Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis. Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPgammaS, indicating a requirement for GTPases and for the vacuolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.