We have analyzed the activities of the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), WR99210, trimethoprim (TMP), methotrexate (MTX), and trimetrexate (TMX) against Kenyan Plasmodium falciparum isolates adapted in vitro for long-term culture. We have also assessed the relationship between these drug activities and mutations in dihydrofolate reductase (dhfr), a domain of the gene associated with antifolate resistance. As expected, WR99210 was the most potent drug, with a median 50% inhibitory concentration (IC50) of <0.075 nM, followed by TMX, with a median IC50 of 30 nM. The median IC50 of CCG was 37.80 nM, and that of MTX was 83.60 nM. PM and TMP were the least active drugs, with median IC50s of 733.26 nM and 29,656.04 nM, respectively. We analyzed parasite dhfr genotypes by the PCR-enzyme restriction technique. No wild-type dhfr parasite was found. Twenty-four of 33 parasites were triple mutants (mutations at codons 108, 51, and 59), and only 8/33 were double mutants (mutations at codons 108 and 51 or at codons 108 and 59). IC50s were 2.1-fold (PM) and 3.6-fold (TMP) higher in triple than in double mutants, though these differences were not statistically significant. Interestingly, we have identified a parasite harboring a mutation at codon 164 (Ile-164-Leu) in addition to mutations at codons 108, 51, and 59. This quadruple mutant parasite had the highest TMP IC50 and was in the upper 10th percentile against PM and CCG. We confirmed the presence of this mutation by sequencing. Thus, TMX and MTX are potent against P. falciparum, and quadruple mutants are now emerging in Africa.