The time of initiation of fluconazole treatment with or without dexamethasone, and the impact on mycological outcome and drug pharmacokinetics were assessed in a murine model of disseminated cryptococcosis. Non-infected mice and mice with disseminated cryptococcosis were given saline, dexamethasone, or fluconazole +/- dexamethasone, 1 or 8 days after infection. Cfus were counted in tissues, and fluconazole concentrations were determined in plasma and tissues by HPLC and a bioassay. Despite fluconazole tissue and plasma concentrations which were above the minimal inhibitory concentration, the numbers of cfus in brain and lung tissues were reduced after early (P = 0.002 and 0.04, respectively), but not after late fluconazole treatment. The administration of dexamethasone did not have a deleterious effect on the number of cfus, fluconazole pharmacokinetics or antifungal activity. In conclusion, the size of the fungal burden influences the effective level of fluconazole activity in lung and brain. These results strongly suggest that potential antifungal agents should be studied following both early and late administration in experimental cryptococcosis.