Osmotic swelling of Trypanosoma cruzi epimastigotes resulted in alkalinization of acidocalcisomes, as revealed by changes in acridine orange fluorescence of intact cells. Concomitant with these changes, intracellular ammonium levels increased while extracellular ammonium levels decreased significantly. Hypo-osmotic stress also resulted in cytosolic acidification. The observed changes in intracellular pH (pH(i)) were independent of extracellular calcium, and other ions concentration. Taken together, these results are consistent with a stimulation of ammonium production upon hypo-osmotic stress and its accumulation in acidocalcisomes resulting in their alkalinization, which might be responsible for polyphosphate hydrolysis and osmotic changes in the organelles.