Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain- containing protein 1 (DFCP1)positive omegasome and the IM accumulated in Atg3- deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)- tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/ or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200- deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.