The codon for serine residue 108 of thePlasmodium falciparumdihydrofolate reductase gene was replaced with those for the other 19 amino acids. Except for the Lys 108 mutant, which was not expressed, all other substitutions yielded DHFR mutants which were expressed inEscherichia colias inactive inclusion bodies. Nine of the mutants—Asn 108 , Thr 108 , Gly 108 , Ala 108 , Gln 108 , Cys 108 , Val 108 , Leu 108 , and Met 108 —yielded active DHFR upon refolding of the protein from the inclusion bodies. The remaining mutants—Ile 108 , Arg 108 Pro 108 , Asp 108 , His 108 , Tyr 108 , Phe 108 , Trp 108 , and Glu 108 —did not exhibit detectable DHFR activity on refolding. The Asn 108 mutant had almost unperturbed kinetic parameters but conferred resistance to pyrimethamine and cycloguanil; other active mutants showed poorer DHFR activity. We purified and characterized four mutants which produced highest DHFR activity, i.e., the Gln 108 , Gly 108 , Cys 108 , and Ala 108 mutants. These mutant enzymes hadk cat /K m values ranging from 7 to 22% of the wild-type enzyme. While DHFRs from Gly 108 , Cys 108 , and Ala 108 mutants were as susceptible to pyrimethamine and cycloguanil as the wild type, the Gln 108 mutation conferred high resistance to both inhibitors. Our data suggest that residue 108 is important for antifolate binding, and that the Ser 108 to Asn 108 mutation was selected in nature because of (i) the need for only a single base change, (ii) its good activity, and (iii) its resistance to antifolates.