Drug resistance is contributing to increasing mortality from malaria worldwide. For assessment of the role of resistance-conferring parasite mutations on treatment responses to sulfadoxine-pyrimethamine (SP) and transmission potential, 120 subjects with uncomplicated falciparum malaria from Buenaventura, Colombia, were treated with SP and followed for 21 days in the period February 1999 to May 2000. Exposures of interest were mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase that confer resistance to pyrimethamine and sulfadoxine, respectively. Although SP was highly efficacious (96.7%), the presence together of DHFR mutations at codons 108 and 51 was associated with longer parasite clearance time (relative hazard = 0.24, p = 0.019) more so than the 108 mutation alone (relative hazard = 0.45, p = 0.188). This association remained after controlling for potential confounders. Infections with these mutations were also associated with the presence of gametocytes, the sexual form of the parasite responsible for transmission, 14 and 21 days after treatment (p = 0.016 and p = 0.048, respectively). Higher gametocytemia is probably due to DHFR mutations prolonging parasite survival under drug pressure, resulting in longer parasite clearance time and allowing asexual parasites to differentiate into gametocytes. These results suggest that even when SP efficacy is high, DHFR mutations that are insufficient to cause therapeutic failure may nevertheless increase malaria transmission and promote the spread of drug resistance.