We reported that a super oxide dismutase mimetic agent, tempol, inhibited elevation of indoxyl sulfate (IS) and recovered impaired vascular function in adenine-induced kidney disease rats. These results suggested that reactive oxygen species (ROS) accelerated IS production or tempol directly inhibited IS production. The aim of study is to clarify the effect of tempol on IS production. We measured IS production by the use of rat liver S9 fraction in vitro. Tempol weakly inhibited hepatic IS production (IC₅₀ of 138 mM). We examined the effect of ROS on IS production by using H₂O₂ or free radical generators, 2,2‘-azobis (2-amidinopropane) dihydrochloride (AAPH). Although H₂O₂ failed to affect IS production, AAPH accelerated IS production. These results suggested that ROS accelerated IS production. In conclusion, tempol seems to inhibit IS production through both directly inhibit sulfotransferase or cytochrome P450 and reduction of ROS. This finding suggests that antioxidant may be useful therapy of cardiorenal syndrome mediated by uremic toxin.