Phosphatases terminate signaling events induced by cytokines and growth factors. Reactive oxygen species (ROS) are considered inhibitors of the activity of protein tyrosine phosphatases. Less is known concerning the inhibitory properties of ROS on the activity of dual specific phosphatases. MAPK-phosphatase-1 (MKP-1) belongs to this family of phosphatases and is of particular importance for cellular function as it dephosphorylates Erk1/2 as well as p38 MAPK and JNK. Besides mitochondria, the NADPH oxidase family is the most prominent source of ROS in many cells. Of the 5 NADPH oxidases (Nox1–5) present in cells, only Nox4 constitutively produces ROS. We found that MKP-1 is a direct effector of Nox4: Overexpression of Nox4 increased MKP-1, whereas siRNA-mediated downregulation of MKP-1 in 3T3-L1cells. Accordingly, in luciferase reporter gene assays Nox4 siRNA reduced the basal activity of the MKP-1 promotor to 75% compared to scr-transfected cells and overexpression of Nox4 enhanced the insulin-inducible MKP-1 promotor activity by 30%. Also the insulin-induced increase in Nox4 expression paralleled the increase in MKP-1 protein expression in these cells. Accordingly, insulin-induced phosphorylation of Erk1/2 was attenuated in 3T3-L1 overexpression Nox4 but was enhanced when Nox4 was depleted by siRNA. Given the notion that ROS could inactivate phosphatases, we studied whether or not Nox4 interferes with the activity and turnover of MKP-1. Inhibition of proteasomal degradation increased MKP-1 protein abundance. Neither Nox4 overexpression nor depletion of Nox4 by siRNA influenced the ubiquitinilation of MKP-1 and thereby its proteasomal degradation. We next studied the effect of Nox4 on MKP-1 activity: An in vitro dephosphorylation assay using phosphorylated Erk1/2 and immunoprecipitated MKP-1 from Hek293 cells expressing different plasmids was used: Overexpression of Nox4 increased ROS production approx. 6fold and importantly Nox4 rather increased then impaired MKP-1 activity. By an alternative approach we analysed the dephosphorylation of the synthetic substrate p-nitrophenyl phosphate (pNPP) using the whole cell-lysate of 3T3-L1 overexpressing MKP-1 or MKP-1 in combination with Nox4 after inhibition of tyrosine-phosphatases. Also in this assay an increase rather than a decrease in phosphatase activity was observed after Nox4 overexpression. As thiol oxidation frequently underlies phosphatase inactivation, we analysed whether or not MKP-1 can be stained by biotinylated iodoacetamid (BIAM). At pH=6,5 BIAM specifically reacts with reduced thiols and at pH=8.5 all thiols are marked. Neither the specific nor the unspecific pH conditions revealed any significant MKP-1 BIAM-labeling. In Conclusion: Nox4-mediated ROS production does not attenuate the activity of MKP-1, possible due to the lack of accessible reduced thiols. Further Nox4 seems to be directly involved in the expression of MKP-1 by controlling the promoter-activity.