We have exploited the experimental accessibility of the protozoan parasite Toxoplasma gondii and its similarity to Plasmodium falciparum to investigate the influence of specific dihydrofolate reductase polymorphisms known from field isolates of drug-resistant malaria. By engineering appropriate recombinant shuttle vectors, it is feasible to examine mutations by transient or stable transformation of T. gondii parasites, in bacterial and yeast complementation assays, and through biochemical analysis of purified enzyme. A series of mutant alleles that mirror P. falciparum variants reveals that the key mutation Asn-108 (Asn-83 in T. gondii) probably confers resistance to pyrimethamine by affecting critical interactions in the ternary complex. Mutations such as Arg-59 (T. gondii 36) have limited effect in isolation, but in combination with other mutations they enhance the competitive ability of folate by increasing the speed of product turnover. Val-16 (T. gondii 10) confers low level resistance to cycloguanil but hypersensitivity to pyrimethamine. This mutation precludes Asn-108, probably because compression of the folate binding pocket introduced by this combination is incompatible with enzyme function. These studies permit detailed biochemical, kinetic, and structural analysis of drug resistance mutations and reconstruction of the probable phylogeny of antifolate resistance in malaria.