We investigated the significance of endogenous reactive oxygen species (ROS) produced by fungi treated with miconazole. ROS production in Candida albicans was measured by a real-time fluorogenic assay. The level of ROS production was increased by miconazole at the MIC (0.125 micro g/ml) and was enhanced further in a dose-dependent manner, with a fourfold increase detected when miconazole was used at 12.5 micro g/ml. This increase in the level of ROS production was completely inhibited by pyrrolidinedithiocarbamate (PDTC), an antioxidant, at 10 micro M. In a colony formation assay, the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of PDTC. Moreover, the level of ROS production by 10 clinical isolates of Candida species was inversely correlated with the miconazole MIC (r = -0.8818; P < 0.01). These results indicate that ROS production is important to the antifungal activity of miconazole.