Ca 2+ is an essential activator of motility in the obligate intracellular parasiteToxoplasma gondii.Ca 2+ ionophore A23187 and intracellular microinjection of Ca 2+ initiate motility of parasites residing in parasitophorous vacuoles (PV). The source of Ca 2+ and the mechanism by which it activates motilityin vivoremain uncertain. Exposure of the parasites to dithiothreitol (DTT) can activate egress of previously nonmotile intravacuolar parasites within 60 sec. DTT is also known to activate both isoforms of the highly concentrated nucleoside triphosphate hydrolase (NTPase) produced byT. gondii.Using an adherent cell analysis system (ACAS) for Ca 2+ imaging, a brief 15–50% increase in intra-PV fluorescence ratio was observed after exposure of infected fibroblasts to 5 mMDTT. Chelation of intracellular Ca 2+ with BAPTA-AM and extracellular Ca 2+ with EGTA blocked the DTT effect; however, this chelation did not prevent the activation of parasites nor the Ca 2+ response to the Ca 2+ ionophore ionomycin, suggesting that the Ca 2+ that activates motility may reside near or within the parasite itself. This result demonstrates that an increase in Ca 2+ within the vacuole precedes the onset of motility and the correlation of the DTT effect on motility and tachyzoite NTPase suggests that NTPase activation may be involved in the Ca 2+ flux.