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Large intracellular vacuoles, >4 μm in diameter and either round or oval-shaped, were observed infrequently in Dictyostelium discoideum amoebae of axenically-grown strain AX2 (only 1 in 106–108 cells). These previously unreported single or multiple “giant” vacuoles were more common, however, in newly germinated KAX3 cells (0.55% of the population) and AT-Kneg, a strain that lacks an esterase (0.47% of the population). A vacuolar H+-ATPase was enriched in their membranes of intracellular giant vacuoles, indicating that the vacuoles were related possibly to both endosomes and the contractile vacuole compartment. When monitored over time, giant vacuoles protruded from, and retracted back into cells under hyperosmotic conditions, suggesting an osmoregulatory role for these vacuoles. Some of the intracellular and protruded giant vacuoles harbored a fluid-phase marker, fluorescein-labeled dextran, implying a pinocytotic origin for the vacuoles.