Accumulating evidence demonstrates that oxidative damage is one of the underlying mechanisms to the cytotoxicity and carcinogenicity of AFB1. The main objective of this study is to show that AFB1 increases reactive oxygen species (ROS) formation in hepatocytes. The ROS level was detected using a fluorescence probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS. It was found that AFB1 exposure significantly enhanced DCF fluorescence formation in cultured rat hepatocytes. A dose-response of AFB1 was also observed within the range of 10 nM to 1000 nM. Catalase (CAT) was able to completely prevent the increase of DCF fluorescence in AFB1-treated cells in a dose-dependent manner (from 500 to 2000 U/ml). Moreover, the significant inhibitory effects of desferrioxamine (DFO) and dimethyl sulfoxide (DMSO) on DCF fluorescence formation were also observed in both control and AFB1-treated hepatocytes. Therefore, results from the present study provide in vitro evidence indicating the generation of ROS in cultured rat hepatocytes caused by AFB1 exposure. It is postulated that the metabolic process of AFB1 by cytochrome P450 might be the possible source of the elevated ROS level in AFB1-treated hepatocytes. The enhanced level of ROS may be responsible for the oxidative damage caused by AFB1, which may ultimately contribute to the cytotoxic and carcinogenic effects of AFB1.