Two distinct populations of acid hydrolase-containing vesicles have been found in developing Dictyostelium discoideum cells. Percoll gradient centrifugation revealed these vesicle populations have densities of 1.07 and 1.13 g/ml. The 1.13 g/ml vesicle populations arose during the aggregation stage of differentiation. Although both vesicle populations contained an array of acid hydrolases, they could be shown to differ by several criteria. Electron micrographs of prespore cells showed they contained two types of vesicles with distinct acid phosphatase-staining patterns. One of these vesicle types appeared identical to the lysosomes found in vegetative cells. The second vesicle type had a morphology similar to that of a previously identified organelle, the prespore vesicle. The prespore vesicle is known to contain spore coat proteins which are exocytosed during the final stages of spore differentiation. The higher density acid hydrolase-containing vesicle population was found to contain spore coat proteins. Electron micrographs of the higher density vesicle population showed the presence of acid phosphatase-staining vesicles with a morphology similar to that of prespore vesicles. These data suggest that the higher density acid hydrolase-containing vesicles represent a subpopulation of lysosomes which appear during development and which may be identical to prespore vesicles.