Autophagy is a highly conserved degradative pathway whereby a double membrane engulfs cytoplasmic constituents to form an autophagic vacuole or autophagosome. An essential requirement for efficient autophagy is the acquisition of an adequate degradative capacity by the autophagosomes. To acquire this capacity the immature autophagic vacuoles (AVis) obtain lysosomal hydrolases by fusion with endosomes. The current models suggest that at least two types of endosomes, early and late, fuse with AVis to form mature, degradative AVds. This fusion and maturation requires proteins also involved in endosome maturation such as Rab7. However, it is not known if there are molecular requirements unique to AVi-endosome fusion. To identify and investigate the molecular requirements of this fusion we developed a cell-free fusion assay based on content mixing, which occurs after fusion of isolated AVis and different endosomal fractions. Our assay shows that isolated AVis can fuse to a similar extent in vitro with both early and late endosomes. Furthermore, fusion between autophagosomes and endosomes requires cytosolic and endosomal proteins, but does not show a nucleotide-dependence, and is partially N-ethylmaleimide sensitive. We also demonstrate that the lipidated form of the autophagosomal protein LC3 is dispensable for this fusion event.