Oxidative stress resulting from unilateral ureteral obstruction (UUO) may be aggravated by increased production of reactive oxygen species (ROS). Previous studies demonstrated increased COX-2 expression in renal medullary interstitial cells (RMIC) in response to UUO. We investigated both in vivo and in vitro the role of ROS in COX-2 induction in rats subjected to UUO and RMIC cells exposed to oxidative and mechanical stress. Rats subjected to 3-day UUO were treated with 2 mechanistically distinct antioxidants, the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) and the complex I inhibitor rotenone (ROT), to interfere with ROS production. We found that UUO-mediated induction of COX-2 in inner medulla was attenuated by both antioxidants. In addition, DPI and ROT reduced tubular damage and oxidative stress after UUO. Moreover, mechanical stretch induce COX-2 and oxidative stress in RMIC. Likewise, RMIC cells exposed to H2O2 as an inducer of oxidative stress showed increased COX-2 expression and activity, both of which were reduced by DPI and ROT. Similarly, ROS production, which was increased following exposure of RMIC cells to H2O2, was also reduced by DPI and ROT. Furthermore, oxidative stress-induced phosphorylation of ERK1/2 and p38 was blocked by both antioxidants, and inhibition of ERK1/2 and p38 attenuated COX-2 induction in RMIC cells. Notably, COX-2 inhibitors further exacerbated the oxidative stress level in H2O2-exposed RMIC cells. We conclude that oxidative stress as a consequence of UUO stimulates the COX-2 expression through activation of multiple MAP kinases and that COX-2 induction may exert a cytoprotective function in RMIC cells.