A novel type of membrane vesicles was formed in vitro from microsomes of Saccharomyces cerevisiae, which carries Dpm1p, an enzyme involved in dolichol-sugar synthesis, but not a typical secretory cargo. While COPII vesicles formed in vitro were sedimentable by centrifugation at 200,000gmax for 15 min, the novel vesicles were not. However, they were sedimented by additional centrifugation at the same speed for 1 h. Immunoelectron microscopy showed that the Dpm1p-containing vesicles had small vesicular/saccular structures of around 40–50 nm in diameter. The addition of glycerol-3-phosphate and oleoyl-CoA, substrates for lipid biosynthesis, significantly enhanced the efficiency of vesicle budding in an ATP-dependent fashion. Dpm1p was localized to lipid droplets as well as endoplasmic reticulum. Fluorescence microscopy further showed that Dpm1p-GFP was present in restricted subregions in isolated lipid droplets. The possibility that the vesicles were intermediates from the endoplasmic reticulum to lipid droplets was examined, and their possible role is discussed.