Three diverse mechanisms have been proposed to explain the antimalarial activity of chloroquine: binding to DNA, binding to hemin, or alkalinizing parasite lysosomes. In order to assess the importance of DNA binding, we have measured the affinity of DNA for chloroquine by equilibrium dialysis using tritiated chloroquine. The dissociation constant of the DNA-chloroquine complex varied from 27 microM to 2.6 mM, depending on the ionic strength of the buffer. Our results suggest that chloroquine binding to DNA is highly dependent on salt concentration. Nevertheless, because of the large number of sites present, binding to parasite DNA may still account for the antimalarial activity of chloroquine.