Reactive oxygen species (ROS) generation by microglia is implicated in neuroinflammation and neurotoxicity, as well as in host defense, cell proliferation and excitatory amino acid release. Recent studies demonstrate that primary microglia preparations not only express the phagocyte NADPH oxidase NOX2, but also the NOX1 and NOX4 isoforms. Here we investigated the relationship between neuroinflammation and NOX isoform expression in the human microglia cell line clone 3 (HMC3). HMC3 cells are typical microglia, as suggested by the constitutive expression of Iba-1 and CD14, and IFN-γ-induced expression of CD11b, CD68 and MHCII. However, the characteristics of NOX isoform expression and ROS generation by HMC3 cells were unexpected. RT-PCR demonstrated abundant expression of NOX4, but almost no NOX2 mRNA. ROS generation was constitutive and appeared predominantly intracellular, as superoxide was detected within intracellular vesicles, while the cell-permeable H2O2 was found in the extracellular space. ROS generation by HMC3 was efficiently suppressed by siRNA directed against NOX4, but not by control siRNA. NOX4 suppression did not alter expression of the microglia-typical genes MHCII, CD68 and CD11b, nor did it affect the expression of iNOS, VEGF or TGF-β. However, there was a marked decrease in IL-6 mRNA. Taken together, we demonstrate a constitutive NOX4-dependent ROS generation in a microglial cell line which leads to expression of IL-6 mRNA. The possibility that microglia could switch from tightly regulated NOX2-dependent ROS generation to constitutive NOX4-dependent ROS generation is of interest for the understanding of the role of microglia in maintaining the balance between neuroprotection and neuroinflammatory damage.