Keratinocytes mount immune responses through the secretion of a variety of inflammatory cytokines, soluble proteins and reactive oxygen species (ROS). However, the role of ROS in keratinocytes in response to allergens and irritants has not yet been elucidated. In this study, we investigated the (i) ROS production; (ii) potential sites of ROS production; (iii) expression of cell surface molecules; (iv) secretion of cytokines; and (v) ROS-dependent protein carbonylation in chemical-treated human keratinocyte cell line (HaCaT) cells. Treatment of HaCaT cells with 2,4-dinitrochlorobenzene (DNCB) and benzalkonium chloride (BKC) increased ROS levels in a time- and dose-dependent manner, as determined with dichlorodihydrofluorescein diacetate (CM-H(2) DCFDA), without reducing cell viability. Potential sources of ROS production were evaluated with pretreatment of diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; rotenone, an inhibitor of the mitochondrial electron transport chain complex or allopurinol, a xanthine oxidase inhibitor. The DNCB-induced ROS was related to both NADPH oxidase and mitochondrial electron transport chain complex. Conversely, BKC-induced ROS was related to NADPH oxidase only. Western blotting using an anti-DNP antibody revealed ROS-dependent protein carbonylation in response to DNCB but not BKC. Both DNCB and BKC increased the secretion of IL-1α from HaCaT cells; however, ROS production as well as other changes, except DNCB-induced secretion of IL-1α, was not inhibited by antioxidants. Although the role of ROS in keratinocytes in response to chemicals was inconclusive, our results suggest that the characteristics of ROS produced by keratinocytes in response to chemicals might differ.