Fluorescein isothiocyanate (FITC)-dextran and pyranine were fed to the social amoeba, Dictyostelium discoideum. These membrane-impermeable, pH-sensitive fluorophores initially entered a approximately equal to neutral endocytic compartment. They encountered maximal acidity (pH approximately equal to 5) about 15 min after ingestion, in what appeared to be digestive vacuoles (lysosomes). The environment of the probes returned to near neutrality by 30 min. At that time, the probes accumulated in a decreasing number of vacuoles of increasing size; ultimately, there were only a small number of vacuoles per cell with diameters of up to 3 microns. The late vacuoles sedimented more rapidly than did proton pumps, acid hydrolases, and recently ingested cargo. Unlike the vacuoles harvested immediately after the cells were fed FITC-dextran, the late vacuoles were not acidified by MgATP in vitro. Egestion of ingested FITC-dextran commenced after a lag of approximately equal to 45 min. A similar lag was observed for the resurfacing of two endocytosed bilayer-intercalated fluorophores. These results suggest that, in Dictyostelium, undigested endocytic cargo accumulates in and is returned to the cell surface through a distinctive compartment of large and nearly neutral post-lysosomal vacuoles. It will be important to determine the degree to which internalized plasma membrane components follow this post-lysosomal pathway.