Recently, we measured a more acid digestive vacuolar pH for drug resistant Plasmodium falciparum [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press; Ursos LMB, Dzekunov S, Roepe PD. Mol Biochem Parasitol 2000;in press]. We suggested this acidification contributes to drug resistance via the profound effects that pH has on the solubility of unpolymerized heme found in the vacuole (ferriprotoporphyrin IX mu oxo dimers). In this report, we measure how FPIX concentration, time, NaCl concentration, and several antimalarial drugs affect FPIX pH dependent solubility. Aggregation is essentially instantaneous below pH 5.3, but at vacuolar pH previously measured for HB3 parasites [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press] can increase to several minutes as NaCl is lowered. As FPIX is decreased, the midpoint of the pH dependent solubility curve shifts to higher values. Addition of antimalarial drugs also increases the midpoint of the pH dependent FPIX solubility curve, with the net shift proportional to the relative affinity of the drug for FPIX. Surprisingly, however, for all drugs tested shifts of essentially identical magnitude are found at all drug: FPIX molar ratios inspected, spanning eight orders of magnitude (to as low as 0.0000001:1). This suggests that changes in pH dependent FPIX solubility by addition of antimalarial drugs is via previously unrecognized drug/FPIX nucleation phenomena. These data could have important implications for understanding the role of previously observed changes in pH(vac) [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press; Ursos LMB, Dzekunov S, Roepe PD. Mol Biochem Parasitol 2000;in press] upon development of antimalarial drug resistance.