The calcium- and arachidonic acid (AA)-binding proteins S100A8 and S100A9 are involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in phagocytes. They are specifically expressed in myeloid cells, and are also found in epithelial cells in various (patho)physiological conditions. We have investigated the consequences of S100A8/A9 overexpression in epithelial cell lines on reactive oxygen species (ROS) generation and downstream signaling. Epithelial carcinoma HeLa cells, which exclusively express Nox2, showed dramatically increased activation of NADPH oxidase by phorbol 12-myristate 13-acetate after S100A8/A9 gene transfection. HaCaT keratinocytes overexpressing S100A8/A9 showed enhanced, transient ROS generation in response to the calcium ionophore A23187 compared to mock-transfected cells. Polymerase chain reaction analysis revealed mRNA transcripts for Nox1, Nox2, and Nox5 in HaCaT keratinocytes. Detailed transfection studies confirmed that NADPH oxidase activities in Nox1- and Nox5-transfected HeLa cells were enhanced after S100A8/A9 gene complementation. Furthermore, mutational analysis revealed that AA binding and Thr113 phosphorylation are important for S100A8/A9-enhanced activation of NADPH oxidase. Nuclear factor-kappaB (NF-kappaB) activation and interleukin-8 mRNA levels were increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-kappaB is a redox-sensitive transcription factor. Because they are expressed in epithelia under specific conditions, S100A8 and S100A9 might be involved in skin pathogenesis by modulating aspects of downstream signaling.