Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT_2A receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT_2A receptor antagonist ketanserin. The peak effect was noted at 5–10 min, and half-maximal stimulation was achieved at 10–30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H₂O₂ and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT_2A receptor → G_q protein → phospholipase C → diacylglycerol → classical PKC → NAD(P)H oxidase → superoxide → superoxide dismutase → H₂O₂ → mitogen-activated extracellular signal-regulated kinase → ERK. These studies demonstrate a role for the 5-HT_2A receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H₂O₂ in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.