Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)alpha, although the events through which TNF alpha induces ROS generation are not well characterized. Here, we report that TNF alpha-induced ROS production was blocked by pretreatment with internalization inhibitor monodansyl cadaverine (MDC). Similarly, a transient expression of a GTP-binding and hydrolysis-defective dynamin mutant (dynamin(K44A)) that had been shown to be defective in internalization significantly attenuated the TNF alpha-induced intracellular ROS production. Importantly, the inhibition of receptor internalization suppressed TNF alpha signaling to mitogen-activated protein kinases (MAPKs) stimulation. Together, our results suggest that receptor internalization is somehow necessary for the TNF alpha-induced ROS generation and subsequent intracellular downstream signaling in non-phagocytes. (c) 2006 Elsevier Inc. All rights reserved.