Three different methods were used to determine the in vitro activities of amphotericin B, ketoconazole, itraconazole, and flucytosine against 30 isolates of different genera of filamentous fungi. MICs were determined visually, with or without agitation, and spectrophotometrically by using a broth microdilution method. For amphotericin B, there was one end point reading criterion (the minimum concentration of antifungal that inhibited 100% of growth), but for azoles and flucytosine there were two (the minimum concentrations that inhibited 50 and 75% of fungal growth, respectively) after 48 and 72 h of incubation. All tests were performed in triplicate. An intraclass correlation coefficient (ICC) was used to evaluate the reproducibility of each of the methods and the correlation among them. The reproducibility of the three methods was very high (ICC of 0.808 to 0.992), particularly in the case of azoles and flucytosine. In general, the degree of reproducibility was highest for azoles and amphotericin B after 72 h of incubation and for flucytosine after 48 h of incubation. The degree of correlation among the three methods was very high (ICC of >0.98) with all of the antifungals under all the conditions tested. The end point reading criteria and the time of incubation affected neither the reproducibility of the methods nor their correlation, and their effect on MICs was statistically significant.