Plasmodium falciparum strains bearing quadruple mutations of dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) at codons 51, 59, 108, and 164 are highly resistant to pyrimethamine (PYR), a diaminopyrimidine, but sensitive to WR99210 (WR), a cycloguanil analog, suggesting different enzyme-inhibitor binding interactions. A combination of these inhibitors to delay the onset of antifolate resistance is proposed. Using error-prone PCR, libraries of random mutants of wild-type PfDHFR and PfDHFR-TS were generated and used to transform Escherichia coli, and transformants were then selected for PYR or WR resistance. Mutants highly resistant to either PYR or WR were also generated from libraries obtained from further random mutagenesis of quadruple mutants (QM) with mutations in PfDHFR or PfDHFR-TS. For reversion mutants carrying altered residues I51N, N108S, and L164I, a further mutation of D54N was required to achieve resistance against WR, but these mutants regained sensitivity to PYR. When a combination of PYR and WR was used, fewer resistant mutants were generated from both mutant libraries using the QM gene templates. The effectiveness of the drug combination in reducing the appearance of resistance mutations is likely due to conflicting requirements for mutations conferring resistance to the two drugs. Thus, a combination of inhibitors from these two drug classes should be effective in impeding the emergence of P. falciparum resistance to antifolates.