We present the first single cell-level analysis of digestive vacuolar pH for representative chloroquine resistant (strain Dd2) versus sensitive (strain HB3) malarial parasites. Human red blood cells harboring intact intraerythrocytic parasites were attached to glass substrate, continuously perfused with appropriate buffer, and pH was analyzed via single cell imaging and photometry techniques. We find that digestive vacuolar pH (pH(vac)) is near 5.6 for HB3 parasites. Surprisingly, we also find that pH(vac) of Dd2 is more acidic relative to HB3. Notably, in vitro pH titration of hematin confirms a very steep transition between soluble heme (capable of binding chloroquine) and insoluble heme (not capable of binding chloroquine, but still capable of polymerization to hemozoin) with a distinct midpoint at pH 5.6. We suggest the similarity between the hematin pH titration midpoint and the measured value of HB3 pH(vac) is not coincidental, and that decreased pH(vac) for Dd2 titrates limited initial drug target (i.e. soluble heme) to lower concentration. That is, changes in pH(vac) for drug resistant Dd2 relative to drug sensitive HB3 are consistent with lowering drug target levels, but not directly lowering vacuolar concentrations of drug via the predictions of weak base partitioning theory. Regardless, lowering either would of course decrease the efficiency of drug/target interaction and hence the net cellular accumulation of drug over time, as is typically observed for resistant parasites. These observations contrast sharply with the common expectation that decreased chloroquine accumulation in drug resistant malarial parasites is likely linked to elevated pH(vac,) but nonetheless illustrate important differences in vacuolar ion transport for drug resistant malarial parasites. In the accompanying paper (Ursos, L. et al., following paper this issue) we describe how pH(vac) is affected by exposure to chloroquine and verapamil for HB3 versus Dd2.