Inner medullary collecting duct (IMCD)-derived endothelin-1 (ET-1) is stimulated by volume expansion, in part through augmented luminal flow, whereupon it can elicit natriuresis and diuresis. Since flow can alter nitric oxide (NO) and reactive oxygen species (ROS), both of which can affect collecting duct salt transport, we asked whether NO and/or ROS mediate flow-stimulated IMCD ET-1. Mouse IMCD3 cells were exposed to flow and ET-1/GAPDH mRNA assessed. A shear stress of 10 dyne/cm2 for 1 hr increased ET-1 mRNA by 4-fold compared to no flow (ET-1 flow response). Global NO synthase (NOS) inhibition (L-NAME) reduced the ET-1 flow response, however pharmacologic inhibition of NOS1 or NOS2, NOS3 siRNA, arginase inhibition, removal of media L-Arg, or inhibition of NO-dependent signaling pathways (PKG, guanylyl cyclase or NFkB) did not affect the ET-1 flow response. Tempol reduced the ET-1 flow response; no further inhibition occurred with L-NAME. Superoxide dismutase, but not catalase, reduced the ET-1 flow response. Inhibition of NAPDH oxidase (NOX) (apocynin), pharmacologic inhibition of NOX1/4, or NOX4 siRNA reduced the ET-1 flow response. Finally, flow increased IMCD3 ROS production and this was inhibited by apocynin, NOX1/4 inhibition and, to a small extent, by L-NAME. Taken together, these data suggest that NOX4-derived ROS in general, and possibly superoxide in particular, are involved in flow-stimulated IMCD ET-1 production. To our knowledge, this is the first report of flow-stimulated ROS production by the CD, as well as the first report of such flow-stimulated CD ROS exerting a biologic effect.