The coordinate fusion of the prespore vesicles (PSVs) with the plasma membrane at the terminal stage of spore differentiation in Dictyostelium discoideum is an important example of developmentally regulated protein secretion. However, little is known about the composition of the vesicles, the molecular signals regulating secretion, or the mechanics of the membrane fusion. Taking a biochemical approach, we purified PSVs from different developmental stages. These preparations are highly enriched for their specific cargo of spore coat proteins while devoid of markers for other cellular compartments. Electron microscopic observations show that the PSV preparations are homogenous, with the soluble spore coat protein PsB/SP85 distributed throughout the lumen and the acid mucopolysaccharide localized in the central core. During development the PSVs increase in size and density concomitant with an increase in their protein cargo. The PSVs contain approximately 80 proteins, and we have identified a PSV-specific GTP-binding protein that may be involved in regulating vesicle fusion. The PSVs are not clathrin-coated and do not contain the SpiA spore coat protein. The PSV preparations are ideal for a global proteome analysis to identify proteins involved in signal reception, vesicle movement, docking, and fusion in this developmentally regulated organelle.