The XTT colorimetric assay quantifies fungal growth by measuring fungal metabolism and has been used successfully for susceptibility testing of Aspergillus species after 24 and 48 h of incubation. In the present study using 14 clinical isolates of Zygomycetes (Rhizopus oryzae Infections caused by Zygomycetes have emerged as an increasingly important cause of morbidity and mortality among severely immunocompromised patients, including those with hematological malignancies and hematopoietic stem cell transplant recipients. The prognosis of zygomycosis in these patients remains extremely poor, despite antifungal treatment (10,16,18,19). For years, amphotericin B has been the drug of choice for these devastating infections. Of the recently introduced second-generation triazoles, voriconazole is not active against the Zygomycetes, while posaconazole has demonstrated activity in vitro, in animal models, and in case reports (4-6, 8, 20-22). As zygomycosis may be rapidly fatal, timely determination of the susceptibilities of clinical isolates to these antifungal agents is important.[A colorimetric method, based on reduction of the tetrazolium salt 2,3-bis{2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) by mitochondrial dehydrogenases, has been recently introduced for in vitro susceptibility testing of filamentous fungi. This method quantifies fungal growth by measuring fungal metabolism, using appropriate concentrations of XTT and an electron transfer agent, such as menadione (1,11,12). For Aspergillus species, MICs determined by the XTT method at 24 and 48 h were comparable with those obtained by the National Committee for Clinical Laboratory Standards (now known as the Clinical and Laboratory Standards Institute [CLSI]) method (12). The sensitivity of the XTT assay may be increased depending on the menadione concentration (11). High menadione concentrations could increase the rate of XTT conversion and theoretically allow the detection of metabolic activity associated with small, early fungal growth that is not yet detected visually or spectrophotometrically. In this case, it is likely that the XTT method might even be useful for early determination of MICs of antifungal agents. This hypothesis has been previously discussed (12) but has not been investigated.In preliminary studies using the XTT assay for zygomycete isolates, high metabolic activity was observed even in the presence of small fungal growth (assessed spectrophotometrically) at 12 or 24 h postinoculation and preceded increases in biomass (C. Antachopoulos, J. Meletiadis, E. Roilides, T. Sein, and T. J. Walsh, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-1003, 2005). The present study investigated whether significant metabolic activity with the XTT assay could be demonstrated for the Zygomycetes before hyphal growth is macroscopically or spectrophotometrically detected (at 6 or 8 h postinoculation) and whether changes in this early metabolic activity in the presence of antifungal agents could be used f...