The autophagic/lysosomal system includes a variety of vesicular compartments that undergo dynamic fusion events. However, the characteristics and factors modulating these interactions remain, for the most part, unknown. To gain insights on the properties that govern lysosomal fusion events, we have established an in vitro fusion assay using different lysosomal/autophagic compartments isolated from mouse liver. We have found that autophagosome/lysosome fusion is a temperature-dependent process (fusion increment of 0.2±0.01%/C), which requires ATP (1–3 mM), GTP (1–2 mM), Ca 2+(0.2–2 mM), and an acidic lysosomal pH (pH 5.2). Furthermore, changes in membrane lipid composition, induced either in vitro, by treatment with 25 mM methyl-β-cyclodextrin, or in vivo, by subjecting animals to a high-fat-diet challenge (60% kcal in fat) reduce autophagosome/lysosome fusion up to 70% of that observed in untreated fractions or from animals under a normal regular diet. These findings reveal a novel role for lipids in autophagic fusion and provide a mechanism for the reduced macroautophagic rates observed during exposure to a chronic lipid challenge. Changes in the intracellular lipid content (ie, metabolic disorders) may thus have pronounced effects on the fusion step of macroautophagy and affect the overall activity of this intracellular proteolytic pathway.—Koga, H., Kaushik, S., Cuervo, AM Altered lipid content inhibits autophagic vesicular fusion.