Pyrimethamine, in combination with sulfadoxine, is currently one of the major alternative drugs used for the treatment of chloroquine-resistant Plasmodium falciparum malaria infections in Africa. The mechanism of pyrimethamine resistance has been strongly associated with a single, key point mutation in the dihydrofolate reductase-thymidylate synthase gene, resulting in the substitution of the wild-type allele Ser-108 by either Asn-108 or Thr-108. The pyrimethamine-resistant phenotype and/or genotype were determined in 273 Cameroonian clinical isolates obtained in Yaounde by in vitro assays and polymerase chain reaction-restriction fragment length polymorphism over a 5-year period. The in vitro assays showed that 42% (18 of 43) and 63% (69 of 110) of the isolates obtained in 1994-1995 and 1997-1998, respectively, were resistant to pyrimethamine (50% inhibitory concentration [IC50] > 100 nM). The polymerase chain reaction showed that 43% (55 of 127) and 59% (50 of 85) of the isolates in 1994-1995 and 1997-1998, respectively, had the mutant Asn-108 allele. The pyrimethamine-resistant genotype (Asn-108) corresponded with the pyrimethamine-resistant phenotype (IC50 > or = 100 nM) in a large majority (> 95%) of the isolates. The results of our study suggest an increasing prevalence of pyrimethamine resistance in Yaounde. Our study further suggests that pyrimethamine resistance can be monitored by a technique that can be adopted by malaria research centers in Africa.