The outcome of central nervous system (CNS) histoplasmosis is often unfavorable. Although fluconazole plays an integral role in treatment of fungal meningitis, its role in the treatment of histoplasmosis is hampered by reduced activity and potential development of resistance. A murine model of CNS histoplasmosis was used to evaluate the hypothesis that a combination of amphotericin B and fluconazole therapy would be superior to amphotericin B monotherapy. Groups of B6C3F(1) mice were infected by injection of Histoplasma capsulatum into the subarachnoid space. The addition of fluconazole hindered the antifungal effect of amphotericin B, as determined by measurement of fungal burden, suggesting antagonism in the brain. Fluconazole was less effective as a single agent than was amphotericin B, despite the greater penetration of fluconazole into brain tissues. The hypothesis that amphotericin B-fluconazole combination therapy would be superior to amphotericin B monotherapy for treatment of CNS histoplasmosis was not supported by this study.