The echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthetizing 1,3-β glucan, an essential compound of the fungal wall. Studies in rodents showed that caspofungin can treat Pneumocystis infections. However, its efficacy against Pneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of the P. jirovecii Gsc1 subunit, as well as of those of Pneumocystis carinii and Pneumocystis murina infecting respectively rats and mice. In absence of an established in vitro culture method for Pneumocystis species, we used functional complementation of the Saccharomyces cerevisiae gsc1 deletant. In the fungal pathogen Candida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into the Pneumocystis gsc1 genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The minimum inhibitory concentrations of caspofungin determined by E-test® and Yeastone® for strains complemented with Pneumocystis enzymes (respectively 0.125 and 0.12 microg/ml) were identical to those upon complementation with the enzyme of C. albicans that is sensitive to caspofungin. However, they were lower than the MICs upon complementation with the enzyme of the resistant species Candida parapsilosis (0.19 and 0.25). Sensitivity levels of Gsc1 enzymes of the three Pneumocystis species were similar. Our results suggest that P. jirovecii is sensitive to caspofungin during infections, as P. carinii and P. murina.