Folate antagonizes the antimalarial action of sulfadoxine-pyrimethamine (SP) in vitro, but its role in vivo is not well understood. We measured blood folate concentrations and SP therapeutic outcomes in Malawian children. Children with late treatment failure and those with adequate clinical and parasitologic responses had similar demographic characteristics, prevalence of parasite mutations conferring resistance to SP, and blood concentrations of anti-malarial drugs following treatment. However, a higher folate concentration was associated with late treatment failure. Patients from a low malaria transmission site had higher blood folate concentrations than those in a higher transmission site (mean +/- SEM = 39 +/- 9.3 ng/mL versus 29 +/- 10 ng/mL; P < 0.0001), and there was a higher rate of late treatment failure in the low transmission area (54.4% versus 40.2%; P = 0.010). This study also provides the first evidence of the independent role of physiologic folate concentrations in in vivo SP therapeutic efficacy, and the critical role of pyrimethamine concentrations in the therapeutic efficacy of SP when one controls physiologic folate levels and the frequency of critical dihydrofolate reductase/dihydropteroate synthase mutations.