Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced V(max) values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.