With emerging drug resistance in Plasmodium falciparum, novel antifolates effective against pyrimethamine-resistant and cycloguanil-resistant dihydrofolate reductase (DHFR) are in demand. Based on structural similarity to cycloguanil, it has been proposed that WR99210, and its metabolic precursor PS-15, exerts selective antimalarial activity by binding tightly to both drug-sensitive and drug-resistant DHFR. In the present study, Linweaver-Burk plots and Ackermann-Potter plots reveal that both forms of malarial DHFR bind WR99210 at subnanomolar concentrations. It is not necessary to invoke an alternate target for WR99210 in P. falciparum. The present studies confirm that malarial DHFRs offer potential binding interactions in the folate-binding pocket distinct from those exploited by pyrimethamine and cycloguanil. These kinetic studies also provide a useful framework for the design and interpretation of future structural studies on drug-resistant DHFR from P. falciparum.