Failure of anti-Pneumocystis jiroveci prophylaxis with sulfa drugs is associated with mutations within the putative active site of the fungal dihydropteroate synthase (DHPS), an enzyme encoded by the multidomain FAS gene. This enzyme is involved in the essential biosynthesis of folic acid. The most frequent polymorphisms are two mutations leading to two amino acid changes ((55)Trp-Arg-(57)Pro to (55)Ala-Arg-(57)Ser), observed as a single or double mutation in the same P. jiroveci isolate. In the absence of a culture method for P. jiroveci, we studied potential resistance to sulfa drugs conferred by these polymorphisms by using Saccharomyces cerevisiae as a model. Single or double mutations identical to those observed in the DHPS domain of the P. jiroveci FAS gene were introduced by in vitro site-directed mutagenesis into alleles of the S. cerevisiae FOL1 gene, which is the orthologue of the P. jiroveci FAS gene. The mutated alleles were integrated at the genomic locus in S. cerevisiae and expressed by functional complementation in a strain with a disrupted FOL1 allele. The single mutation (55)Trp to (55)Ala conferred resistance to sulfanilamide, whereas the single mutation (57)Pro to (57)Ser conferred resistance to both sulfanilamide and sulfadoxine. Both single mutations also separately conferred hypersensitivity to sulfamethoxazole and dapsone. The resistance to sulfadoxine is consistent with epidemiological data on P. jiroveci. The double mutation (55)Trp-Arg-(57)Pro to (55)Ala-Arg-(57)Ser conferred on S. cerevisiae a requirement for p-aminobenzoate, suggesting reduced affinity of DHPS for this substrate. This characteristic is commonly observed in mutated DHPS enzymes conferring sulfa drug resistance from other organisms. However, the double mutation conferred hypersensitivity to sulfamethoxazole, which is not in agreement with epidemiological data on P. jiroveci. Taken together, our results suggest that the DHPS polymorphisms observed in P. jiroveci confer sulfa drug resistance on this pathogen.