The in vitro activity of itraconazole was determined against 7,299 isolates of Candida spp., 1,615 isolates of Cryptococcus neoformans, and 445 isolates of Aspergillus spp. obtained from over 200 medical centers worldwide. Itraconazole was active against all Candida spp. (96% of MICs were ≤1 μg/ml) with the exception of C. glabrata (77% of MICs were ≤1 μg/ml). Itraconazole inhibited 94% of C. krusei and 84% of other fluconazole-resistant Candida species, exclusive of C. glabrata, at a MIC of ≤1 μg/ml. Itraconazole was not active against fluconazole-resistant isolates of C. glabrata. Only modest activity was seen against C. neoformans (80% of MICs were ≤1 μg/ml); however, itraconazole showed excellent activity against Aspergillus spp. (94% of MICs were ≤1 μg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.