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A total of 70 Plasmodium falciparum isolates were tested in vitro against pyrimethamine (PYR), tri-methoprim (TRM), sulfadoxine (SDX), and sulfamethoxazole (SMX), and their dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genotypes were determined. dhfr genotypes correlated with PYR and TRM drug responses (r = 0.93 and 0.85). Isolates with wild-type alleles showed mean half inhibitory concentrations (IC50 ± SD) of 0.10 ± 0.10 and 0.15 ± 0.06 μg/100 μl for PYR and TRM. Parasites with mutations at codons 108 and 51 alone or combined with codon 59 have IC50 of 11.46 ± 0.86 (PYR) and 2.90 ± 0.59 μg/100 μl (TRM). For both drugs, the differences in the mean IC50 between wild and mutant parasites were statistically significant (P < 0.001). Isolates with mixed wild and mutant alleles showed an intermediate level of susceptibility. Our data show partial cross-resistance between PYR/TRM and SDX/SMX (r = 0.85 and 0.65). Correlation was not observed between different dhps genotypes and the in vitro outcome to SDX and SMX (r = 0.30 and 0.34). The lack of correlation could be due to folates and para-aminobenzoic acid in the RPMI medium and the serum used to supplement the cultures.