Our studies on endocytosis in Entamoeba histolytica trophozoites suggest that there are two vacuolar compartments in this organism. The first compartment consists of large vacuoles (greater than 2 microns diameter). As measured by the fluid phase markers, fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP), this compartment is a rapid equilibrium with the external milieu and is constantly exchanging (1-2 h) its contents with the external medium. The contents of these vacuoles are not acidified. This together with the absence of degradation of fluid phase markers clearly differentiates these vacuoles from lysosomes of eucaryotes. By labeling externally disposed peptides on the surface membrane of trophozoites with 125I, we could show that the surface membrane was rapidly internalized over a 2-h period and then reached a plateau. All major 125I surface proteins, with the exception of a set of peptides in the 40,000 molecular weight range, were interiorized and approximately 60% of the total radiolabel were found to be in the internal membrane fraction at any given time. The kinetics of this process were similar to those for the uptake of fluid phase markers and are best explained by cycling of the surface membrane into the vacuolar compartment(s) and then back to the cell surface. The second vacuolar compartment consisted of small vesicles (less than 2 microns diameter) with acidified contents as indicated by acridine orange uptake. The endocytic nature of these vesicles was shown by their slow (days) labeling with FITC-dextran, and spectral analysis of internalized FITC-dextran confirmed that this second compartment is acidified (pH 5.2).