Chloroquine (CQ), a 4-aminoquinoline, accumulates in acidic digestive vacuoles of the malaria parasite, preventing conversion of toxic haematin to beta-haematin. We examine how bis 4-aminoquinoline piperaquine (PQ) and its hydroxy-modification (OH-PQ) retain potency on chloroquine-resistant (CQ-R) Plasmodium falciparum. For CQ, PQ, OH-PQ and 4 and 5, representing halves of PQ, beta-haematin inhibitory activity (BHIA) was assayed, while potency was determined in CQ-sensitive (CQ-S) and CQ-R P. falciparum. From measured pK(a)s and the pH-modulated distribution of base between water and lipid (logD), the vacuolar accumulation ratio (VAR) of charged drug from plasma water (pH 7.4) into vacuolar water (pH 4.8) and lipid accumulation ratio (LAR) were calculated. All agents were active in BHIA. In CQ-S, PQ, OH-PQ and CQ were equally potent while 4 and 5 were 100 times less potent. CQ with two basic centres has a VAR of 143,482, while 4 and 5, with two basic centres of lower pK(a)s have VARs of 1287 and 1966. In contrast PQ and OH-PQ have four basic centres and achieve VARs of 104,378 and 19,874. This confirms the importance of VAR for potency against CQ-S parasites. Contrasting results were seen in CQ-R. 5, PQ and OH-PQ with LARs of 693; 973,492 and 398,118 (compared with 8.25 for CQ) showed similar potency in CQ-S and CQ-R. Importance of LAR for potency against CQ-R parasites probably reflects ability to block efflux by hydrophobic interaction with PfCRT but may relate to beta-haematin inhibition in vacuolar lipid.