Amphotericin B and fluconazole are current acceptable therapies for cryptococcal meningitis; however, their effect remains suboptimal. The combination of fluconazole and flucytosine has yielded encouraging clinical results in human immunodeficiency virus patients with cryptococcal meningitis. To investigate the biological basis of this finding, we performed in vitro combination testing of fluconazole and flucytosine against 50 clinical strains of Cryptococcus neoformans var. neoformans. Synergy (fractional inhibitory concentration index of < 1.0) was observed in 62% of cases, while antagonism (fractional inhibitory concentration index of > 2.0) was not observed. For cases in which synergy was not achieved (autonomous or additive effects), the beneficial effect of the combination was still seen (i.e., there was still a decrease, although not as dramatic, in the MIC of one or both drugs when used in combination). The in vitro inhibitory action of flucytosine was greatly enhanced by the addition of fluconazole; the flucytosine MICs for Cryptococcus isolates were markedly decreased to concentrations which were severalfold lower than the achievable cerebrospinal fluid flucytosine concentration. On the other hand, the addition of flucytosine did not greatly enhance the in vitro activity of fluconazole if the initial fluconazole MIC for the isolate was > or = 8 micrograms/ml. Controlled clinical studies are warranted to further elucidate the potential utility of fluconazole-flucytosine combination therapy.