Abstract: During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.