The development of the malaria parasite Plasmodium falciparum in human red blood cells induces parasite-dependent perturbations in the permselectivity properties of the host cell membrane. The changes appear as parasites develop from ring to the trophozoite stage and persist during schizogony. In the present work we assessed the permeability changes of the infected cells to anionic substances by the use of radioactive and fluorescent probes. Our data show that i) covalent binding probes, such as diisothiocyano ditritiostilbene disulfonic acid [3H]H2DIDS, which are virtually impermeant to normal red blood cells, became markedly permeant to trophozoites and schizonts, as evidenced by high labeling of intracellular hemoglobin; ii) permeation of the fluorescent anion transport substrate NBD-taurine, measured in the efflux mode, was very rapid and substantially enhanced in parasitized erythrocytes, as compared with noninfected cells; iii) this efflux could not be blocked by H2DIDS, which is a specific inhibitor of anion transport in normal red blood cells; iv) permeation of anionic probes was temperature dependent (Ea:11 +/- 1 kcal/mole); and v) could be blocked by nonspecific transport inhibitors that are known to interact with membrane lipids. The appearance of a new permeation pathway in the host cell membrane of trophozoites is associated with structural modification of the host cell membrane matrix.