Synthetic oligonucleotides and their chemical modifications have been shown to inhibit viral and cellular gene expression by sequence-specific antisense hybridization to target mRNAs. We now report that oligodeoxynucleotide phosphorothioates and their nuclease-resistant modifications are effective in micromolar and submicromolar concentrations against the growth of both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum in vitro. Parasitized human erythrocytes were found to be accessible to radioactively labeled oligodeoxynucleotides, whereas the uninfected erythrocytes did not permit any cellular entry of the same compounds. The dihydrofolate reductase-thymidylate synthase gene of P. falciparum was demonstrated to be a good target for sequence-dependent inhibition of plasmodial growth by exogenously administered modified oligonucleotides. The antimalarial activities observed in vitro were identical for chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. The antimalarial activity of oligodeoxynucleotide phosphorothioates is related to sequence complementarity to certain regions of the plasmodial genome as well as to non-sequence-defined activities.