bStandardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist for Candida species and Cryptococcus yeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease.T he increasing incidence of fungal disease necessitates adequate and timely assessment of antifungal susceptibility to guide the selection and implementation of antifungal therapies. Consequently, the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have established reference test methods for susceptibility to the major classes of antifungals, which include the polyenes, azoles, and the recently developed echinocandins (1, 2). These standards utilize broth macrodilution or microdilution assays for testing yeasts (M27-A3 [3] and E.DEF 7.1 [4]) and filamentous fungi (M38-A2 [5] and E.DEF 9.1 [6]). Procedures, including inoculum preparation, media, assay duration, and endpoint definitions, have now been established for testing some of the more commonly encountered yeast-form pathogenic fungi (i.e., Candida species and Cryptococcus). However, notably missing from the M27-A3 and E.DEF 7.1 standards for testing of yeasts are methods for testing the yeast forms of the dimorphic fungi.The dimorphic fungi that cause systemic disease are characterized by distinct yeast and filamentous morphological states (7,8). Temperature is the principal morphology-determining factor, with yeast forms characterizing infections in mammals (9). Histoplasma capsulatum is the most common clinically encountered dimorphic fungal pathogen in the United States, with an estimated 10,000 to 20,000...