Pyrimethamine analogs were examined as potential agents against vivax malaria using a bacterial surrogate system carrying Plasmodium vivax dihydrofolate reductase-thymidylate synthase (PvDHFR-TS), in which the PvDHFR complemented chemically knocked out host dihydrofolate reductase. The system was initially tested with P. falciparum dihydrofolate reductase-thymidylate synthase and was found to have good correlation with the parasite-based system. The 50% inhibitory concentrations derived from PvDHFR-TS-dependent bacteria were correlated with their corresponding inhibition constants (K i ) from an enzyme inhibition assay, pointing to the likelihood that the potent enzyme inhibitors will also have potent antimalarial activities. Active compounds against both wild-type and S58R S117N (SP21) double-mutant P. vivax include analogs with structures which can avert a steric clash with the asparagine (S117N) side chain of the mutant, similar to those found for homologous Plasmodium falciparum mutants, raising the possibility that the same compounds can be developed against both types of antifolate-resistant malaria. This rapid and convenient drug screening system should be useful for development of new antifolates against P. vivax, for which a continuous culture system is not yet available.Malaria caused by Plasmodium vivax is a major public health problem in Asia and South and Central America, where it is most prevalent, with estimates of more than 70 to 80 million cases annually (23). The recent reports on a P. vivax parasite resistant to chloroquine (3,20), the drug commonly prescribed for P. vivax infection, in addition to the lack of a protective vaccine, highlight the need for new approaches to antimalarial chemotherapy. One promising drug target for the treatment of P. vivax infections is dihydrofolate reductase (DHFR), a key enzyme in folate biosynthesis and utilization. Antifolates, such as pyrimethamine (Pyr), targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) of the parasite, have been exploited against chloroquine-resistant Plasmodium falciparum, especially as components of combination drugs (2, 9). Nonetheless, these drugs are not recommended for P. vivax treatment due to the preliminary observation that antifolates were ineffective and that the parasite is inherently resistant against them owing to predisposed mutations in the dhfr-ts gene (18,26). Recently, point mutations of DHFR were revealed to have an association with antifolate resistance in P. vivax in vitro (6,8,10,13), leading to the conclusion that P. vivax is initially sensitive to antifolates, and resistance developed through mutations, similar to the case of P. falciparum. This is an important finding on the molecular basis for drug resistance in P.vivax that gives rise to opportunities for effective drug design for P. vivax therapy.Several different methods for assessing antimalarial drug sensitivity have been developed (17). These methods mostly rely on culturing malaria parasites (16,19,25). Unlike the case for P. falciparum, an...