The combination of flucytosine and amphotericin B is first choice treatment for active cryptococcosis. Because of innate or acquired resistance of Cryptococcus neoformans to flucytosine, in vitro testing is mandatory. Yeast nitrogen base (YNB) at pH 7.0 is the recommended medium for the broth microdilution test (NCCLS M27-A) and for the E-test. In order to verify if minimum inhibitory concentrations (MICs) were able to predict treatment outcome, the susceptibility of 24 isolates from 21 patients treated with flucytosine alone or in combination was tested by the broth microdilution, agar dilution and E-test using YNB either at pH 7.0 or at pH 5.4. Only those MICs obtained on YNB pH 5.4 proved to correlate with treatment outcome. The present study suggests that in vitro susceptibility to flucytosine of C. neoformans isolates should be evaluated on YNB pH 5.4 and the test should be standardized accordingly.