The acidic food vacuole exerts several important functions during intra-erythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several antimalarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite’s food vacuole using X-ray microanalysis and quantitative confocal live-cell Ca2+ imaging. Apparent free Ca2+ concentrations of ~90 nM, ~350 nM and ~400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+ sensitive fluorochromes in a live-cell confocal setting. We found that the ratiometric Ca2+ indictor Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.