Reported are (i) the in vitro sensitivities to pyrimethamine of 58 primary Plasmodium falciparum isolates collected from the foothill and coastal areas in Madagascar, and (ii) the results of the amplification of the dhfr gene followed by restriction enzyme digestion of codon 108. Testing took place between March 1999 and February 2000 and all the isolates were sensitive to pyrimethamine. The 50% inhibitory concentration (IC50) ranged from 0.1 nM to 242.9 nM (mean IC50 = 30.5 nM, median IC50 = 4.8 nM, 95% confidence interval 17.6-43.4 nM). None of the 58 isolates yielded digestion products after polymerase chain reaction product treatment with BsrI or ScrFI. The results indicate the absence of the dhfr encoding Asn108 and Thr108 among tested clinical isolates. The sensitivity of P. falciparum to pyrimethamine-based antimalarial drugs in Madagascar is discussed.