Autophagy is an intracellular degradation process, through which cytosolic materials are delivered to the lysosome.Despite recent identification of many autophagy-related genes, how autophagosomes are generated remains unclear.Here, we examined the hierarchical relationships among mammalian Atg proteins. Under starvation conditions, ULK1,Atg14, WIPI-1, LC3 and Atg16L1 target to the same compartment, whereas DFCP1 localizes adjacently to these Atgproteins. In terms of puncta formation, the protein complex including ULK1 and FIP200 is the most upstream unit and is required for puncta formation of the Atg14-containing PI3-kinase complex. Puncta formation of both DFCP1 and WIPI-1 requires FIP200 and Atg14. The Atg12-Atg5-Atg16L1 complex and LC3 are downstream units among these factors. The punctate structures containing upstream Atg proteins such as ULK1 and Atg14 tightly associate with the ER, where the ER protein vacuole membrane protein 1 (VMP1) also transiently localizes. These structures are formed even when cells are treated with wortmannin to suppress autophagosome formation. These hierarchical analyses suggest that ULK1, Atg14 and VMP1 localize to the ER-associated autophagosome formation sites in a PI3-kinase activity-independent manner.