Chloroquine- 14 C was used to study the processes which concentrate chloroquine in mouse red blood cells infected with chloroquine-sensitive or with chloroquine-resistant Plasmodium berghei . The initial rates of uptake and exchange of chloroquine- 14 C were both too fast to measure, yet large concentration gradients were maintained by the cells. When red blood cells were exposed to 10 -8 M chloroquine at 22°C, with pH between 7.2 and 7.4, steady-state gradients of chloroquine- 14 C were approximately 600:1 (cells:medium) for cells infected with chloroquine-sensitive parasites, 100:1 for cells comparably infected with chloroquine-resistant parasites, and 14:1 for uninfected cells. The processes responsible for these gradients were saturable, in agreement with the proposal of chloroquine binding to cellular constituents. No degradation of chloroquine was detected. The major difference between the chloroquine-sensitive and -resistant parasites was deficiency of high-affinity binding of chloroquine by cells infected with chloroquine-resistant parasites. This deficiency explains the reduced ability of chloroquine-resistant parasites to concentrate chloroquine, and it suggests that chloroquine resistance is due to a decrease in the number, affinity, or accessibility of chloroquine receptor sites on a constituent of the malaria parasite.