n Colpoda cucullus, the signaling pathways for encystment induction involving protein phosphorylation have been believed to be triggered by an increase in the intracellular Ca2+ concentration promoted by cell-to-cell mechanical contact due to overpopulation. By means of fura 2 ratiometry, the present study showed that the intracellular Ca2+ concentration was actually elevated when vegetative cells were induced to encyst by being suspended at a high cell density in the presence of external free Ca2+ and suppressed by chelating external Ca2+. This result strongly suggests that an increase in the intracellular Ca2+ concentration caused by an inflow of Ca2+ promoted by cell-tocell mechanical contact due to overpopulation enhances the rate of encystation in Colpoda cucullus.