Entamoeba histolytica is an invasive enteric protozoan parasite whose cytolytic activity is associated with irreversible increases in target cell intracellular calcium. We studied the effect of homogenates of virulent E. histolytica on calcium permeability of bovine chromaffin granules and rat liver mitochondria, model membrane systems whose mechanisms of ion transport and permeability are well characterized. Treatment of chromaffin granules, with an extract of E. histolytica resulted in a dose-dependent increase in calcium uptake. These effects were similar to that seen with the calcium ionophore A23187 and indicate that the homogenate acts as a divalent cation ionophore. However, unlike what is observed with A23187, the Ca2+ uptake was greater in the presence of the permeable anion Cl- than in its absence. Also, in contrast to the calcium ionophore A23187, a homogenate of E. histolytica caused no calcium release from rat liver mitochondria but resulted in a dramatic increase in the rate of calcium accumulation. The amebic homogenate did not bind calcium in the absence of mitochondria. The increase in mitochondria calcium uptake occurred only in the presence of respiration and with normal state 3 and 4 oxygen consumption. Calcium accumulation occurred both in the presence and absence of 5 mM Pi. Substitution of NaCl or KCl for sucrose in the medium did not alter the enhanced mitochondria calcium uptake. Most data are consistent with the hypothesis that the E. histolytica homogenate is acting as a calcium ionophore transporting charged calcium electrophoretically across the chromaffin granules and the mitochondria inner membrane. This activity may contribute to the cytopathogenicity by E. histolytica.